Synthesis and Hybridizing Property of Oligonucleotides Including 2′-C,4′-C-Ethyleneoxy-Bridged 2′-Deoxyadenosine with an Exocyclic Methylene Unit

Content Provider	Semantic Scholar
Author	Hari, Yoshiyuki Osawa, Takashi Onishi, Yoshinori Ito, Yuta
Copyright Year	2020
Abstract	2′,4′-Bridged nucleic acids (2′,4′-BNAs) are of interest because oligonucleotides that include them have excellent duplex-forming capability and high nuclease resistance compared to natural oligonucleotides. We have recently developed 2′-C,4′-C-ethyleneoxy-bridged thymidine with an exocyclic methylene unit (methylene-EoDNA-T) as a novel 2′,4′-BNA analog. Oligonucleotides that include methylene-EoDNA-T have marked hybridizing capability, nuclease resistance, and in vitro gene-silencing potency. In the present study, we designed and synthesized a 2′-deoxyadenosine analog of methylene-EoDNA (methylene-EoDNA-A), and incorporated it into oligonucleotides. The results of melting temperature (Tm) analysis of duplexes formed from methylene-EoDNA-A-modified oligonucleotides indicated that the hybridizing capability with regard to complementary DNA was almost the same or slightly higher than that of natural DNA. Moreover, methylene-EoDNA-A:methylene-EoDNA-T base pairs increased the thermal stability of DNA duplexes compared to that of DNA duplexes containing methylene-EoDNA-Aor methylene-EoDNA-T-modification in one strand. INTRODUCTION Oligonucleotides have been used in gene diagnosis, therapeutic agents, and nanotechnologies.1–7 However, the use of natural DNA or RNA for oligonucleotide-based technologies is problematic because they generally lack binding affinity with complementary single strands and are easily degraded by nucleases. Numerous chemically modified oligonucleotides have been developed to address these issues. Of these, conformationally constrained oligonucleotides—including modified nucleotides with bicyclic carbohydrate moieties—improve the stability of duplexes formed from complementary single strands. In particular, oligonucleotides modified by 2′,4′-bridged nucleic acids (2′,4′-BNAs)8,9/locked nucleic acids (LNAs)10,11 have received significant attention because LNA-modification provides good duplex-forming capability. Moreover, LNA-modified oligonucleotides have improved nuclease resistance owing to the steric hindrance of the bridge moiety. Consequently, various LNA derivatives have been synthesized with the objective of producing an ideal material for practical oligonucleotides.2, 12–14 We recently developed a methylene-EoDNA-T molecule with a six-membered bridge comprising a 6′-oxygen atom and an 8′-exocyclic methylene group (Figure 1).15 The methylene-EoDNA-modified oligonucleotides had excellent hybridizing affinity with complementary RNA, and extremely high resistance to nucleases compared to natural oligonucleotides. Furthermore, the in vitro gene-silencing potency of methylene-EoDNA-T-modified oligonucleotides is comparable to that of LNA-modified oligonucleotides, which have already been used in therapeutic applications.16 However, only thymidine analogs can restrict the application, other nucleoside analogs are required. On the other hand, duplexes including LNA:LNA base pairs are exceptionally stable compared to other duplexes.17 For example, the Tm value of a fully LNA-modified 9-mer duplex was more than 60 °C higher than that of the corresponding natural DNA duplex. Moreover, oligonucleotides modified by 2′,4′-BNANC or 2′,4′-BNACOC, which are 2′,4′-BNA analogs, can form markedly stable homoduplexes.18,19 Therefore, we designed a 2′-deoxyadenosine analog of methylene-EoDNA (methylene-EoDNA-A, Figure 1), because we became interested in the stability of duplexes that include base pairs formed between methylene-EoDNA-A and methylene-EoDNA-T. Herein, we describe the synthesis of methylene-EoDNA-A and the evaluation of the effect of the methylene-EoDNA-T:methylene-EoDNA-A base pair on the stability of DNA duplexes. Figure 1. Structures of methylene-EoDNA-T and methylene-EoDNA-A RESULTS AND DISCUSSION The synthesis of methylene-EoDNA-A began with olefin 2,20 prepared in two steps from adenosine 1 (Scheme 1). After protecting the 2′and 3′-OH groups in compound 2 in situ with trimethylsilyl (TMS) groups, we acylated the NH2 group at the C6 position of adenine with two equivalents of benzoyl chloride, then treated the product with aqueous ammonia to produce N6-benzoyladenosine derivative 3 at a yield of 55% in three steps from 2. The 3′-OH group in 3 was protected by a TBS group to give 5 at a yield of 51%, although 2′-O-TBS compound 4 was also obtained (23% yield). Epoxidation of 5 by in situ-generated dimethyldioxirane using Oxone® and acetone followed by ZnCl2-mediated propargyloxylation at the C4′ position produced the desired 4′-C-propargyloxyadenosine 6 (44% yield) and -lyxofuranosyl derivative 7 (3% yield). Scheme 1. Synthesis of 4′-C-propargyloxyadenosine derivative 6 After protecting the 5′-OH group of diol 6 using DMTrOTf,21 we converted compound 8 to radical precursor 9 using 1,1′-thiocarbonyldiimidazole (TCDI), as shown in Scheme 2. Intramolecular radical cyclization using AIBN and (Me3Si)3SiH afforded the desired cyclized product 10 in 70% yield. Methylene-EoDNA-A-phosphoramidite 12, which is a building block for automated DNA synthesis, was synthesized by the removal of the TBS group in 10 using TBAF followed by phosphitylation of the obtained 11 using (i-Pr)2NP(Cl)O(CH2)2CN. Oligonucleotide synthesis was carried out on an automated DNA synthesizer using common phosphoramidite chemistry with a prolonged coupling time of 10 min (cf. 32 s for coupling of natural phosphoramidites) for the introduction of methylene-EoDNA-A 12 and methylene-EoDNA-T 1315 (Figure 2). To avoid the decomposition of the exocyclic methylene group, 1 M t-BuOOH in toluene,22 instead of 0.02 M iodine solution, was used as an oxidizing agent in oligonucleotide synthesis. Under these conditions, all the desired oligonucleotides (ON1–7, Figure 4) were obtained successfully. Scheme 2. Synthesis of methylene-EoDNA-A phosphoramidite 12 Figure 2. Structure of methylene-EoDNA-T phosphoramidite 13 We evaluated the hybridization properties of methylene-EoDNA-Aand methylene-EoDNA-T-modified oligonucleotides ON1–7 with regard to complementary DNA by UV-melting experiments, and compared the results to those obtained with natural DNA duplexes (ON8 and ON9). Representative UV-melting profiles are shown in Figure 3, and the results of our experiments are summarized in Figure 4. The duplex-forming capability of the methylene-EoDNA-T-modified oligonucleotides ON4–7 (duplexes 4–7, ΔTm/mod. values ranged from −1 °C to +2 °C) was similar to or slightly higher than that of the natural oligonucleotide (ON9, Tm = 48 °C). These results are similar to those of our previous study on the duplex-forming capability of homopyrimidine 14-mer oligonucleotides, including those with methylene-EoDNA-T-modifications (ΔTm/mod. ranged from −2 °C to +1 °C).15 Methylene-EoDNA-A-modified ON1–3 (duplexes 1–3, ΔTm/mod. values ranged from +1 °C to +2 °C) produced similar results to those for methylene-EoDNA-T-modified ON4–7 (duplexes 4–7). We carried out Tm analysis of duplexes 8–11 to evaluate the thermal stability of DNA duplexes containing methylene-EoDNA-A and methylene-EoDNA-T modifications in each strand. Incorporation of a single base pair formed between methylene-EoDNA-A and methylene-EoDNA-T into a DNA duplex (duplex 8) resulted in a ΔTm/mod. value of +0.5 °C, and a methylene-EoDNA-A:methylene-EoDNA-T base pair produced no significant stabilization. Moreover, duplex 9 (ΔTm/mod. = +1 °C), which includes methylene-EoDNA-A and methylene-EoDNA-T modification in each strand, did not exhibit increased stability relative to duplex 1 (ΔTm/mod. = +1 °C) or duplex 5 (ΔTm/mod. = +1 °C). A molecular thermodynamic study of duplexes with LNA-modifications implied that the additional stabilizing effect of LNA-modifications in both strands depends on the location of the two oppositely oriented LNA:DNA base pairs in the duplex.23 Similarly this LNA study, the thermal stability of duplexes including methylene-EoDNA modifications in both strands might be affected by the positions of the modifications in the oligonucleotides, although further investigation is required. Interestingly, duplexes 10 and 11, which contain two methylene-EoDNA-A:methylene-EoDNA-T base pairs per duplex, exhibited synergistically increased stability compared to duplex 8, which includes a single methylene-EoDNA base pair. For instance, the ΔTm/mod. value of duplex 11 was +2.5 °C, which was significantly higher than that of duplex 8 (ΔTm/mod. = +0.5 °C). The synergistic stabilization resulting from the introduction of multiple methylene-EoDNA base pairs has also been observed in duplexes containing multiple 2′,4′-BNANC:2′,4′-BNANC base pairs.18 Moreover, DNA duplexes that have been fully modified by LNA,17 2′,4′-BNANC,18 or 2′,4′-BNACOC,19 have markedly high melting temperatures (Tm > 93 °C). The highly stabilizing effect on the duplex of multiple methylene-EoDNA base pairs might be a general effect of 2′,4′-BNA analogs with constrained sugar moieties that restrict the sugar conformation to a suitable form for duplex formation. Figure 3. The representative data for UV-melting analysis of the modified oligonucleotides Figure 4. ΔTm/mod. values of DNA duplexes formed by methylene-EoDNA-modified oligonucleotides (ON1–7). Conditions: 10 mM sodium cacodylate buffer (pH 7.4), 100 mM NaCl, and 2.5 μM of each oligonucleotide. ΔTm/mod.: The change in Tm value per modification compared with natural DNA duplex (ON8 and ON9, Tm = 48 °C). CONCLUSIONS In the present study, we synthesized the phosphoramidite of methylene-EoDNA-A and incorporated it into oligonucleotides. The results of the UV-melting experiments indicated that the thermal stability of a DNA duplex formed from methylene-EoDNA-Aand methylene-EoDNA-T-modified oligonucleotides was similar to or slightly higher than that of natural DNA. As a result of the evaluation of the stability of DNA duplexes including methylene-EoDNA-A:methylene-EoDNA-T base pairs, it seems that the incorporation of two methylene-EoDNA base pairs significantly increases the
Starting Page	284
Ending Page	284
Page Count	1
File Format	PDF HTM / HTML
DOI	10.3987/com-19-s(f)24
Alternate Webpage(s)	https://heterocycles.jp/newlibrary/downloads/PDF/26287/101/0
Alternate Webpage(s)	https://doi.org/10.3987/com-19-s%28f%2924
Volume Number	101
Language	English
Access Restriction	Open
Content Type	Text
Resource Type	Article