Hybridization and Mismatch Discrimination Abilities of 2â²,4â²-Bridged

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Hybridization and mismatch discrimination abilities of 2#,4#-bridged nucleic acids bearing 2-thiothymine or 2-selenothymine nucleobase Takaki Habuchi, Takao Yamaguchi, Hiroshi Aoyama, Masahiko Horiba, Kosuke Ramon Ito, and Satoshi Obika J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b02863 • Publication Date (Web): 11 Jan 2019 Downloaded from http://pubs.acs.org on January 14, 2019

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The Journal of Organic Chemistry

Hybridization and mismatch discrimination abilities of 2′,4′-bridged nucleic acids bearing 2-thiothymine or 2-selenothymine nucleobase Takaki Habuchi, Takao Yamaguchi, Hiroshi Aoyama, Masahiko Horiba, Kosuke Ramon Ito, Satoshi Obika* Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan Supporting Information Placeholder mismatch discrimination ability X = O 95%, according to the trityl monitor. After the oligomer elongation reaction, the solid support and all protecting groups except for the trityl group were removed using ammonium hydroxide (rt for 1.5 h, then 55 °C for 3 h). Finally, removal of the trityl group under acidic conditions (0.5% trifluoroacetic acid in water) afforded ON1 and ON3 in 38% and 41% yields, respectively. Prolonged ammonia treatment for ON1 (e.g., rt for 1.5 h, then 55 °C for 25 h) resulted in the considerable production of an undesired ON, which was presumably generated by the conversion of the 2thiothymine moiety to 5-methylisocytosine, as previously described by Connolly and Newman.27 ON2 and ON4, even after

2′,4′-BNA/LNA-Se T

5′-d(GCGTTXTTTGCT)-3′

Duplex-forming ability. Next, we evaluated the duplexforming abilities of ON1–ON4 with ssDNA (Table 2). With the full complementary strand (Y = A), ON1–ON4 showed similar to slightly higher UV-melting temperatures (Tm values) compared to that of the natural congener (ON5), revealing that these modified ONs formed regular duplexes. Compared to those of scpBNA-T-modified ON6 and 2′,4′-BNA/LNA-Tmodified ON7,10 ON1 and ON3 had slightly higher Tm values (+1 and +2 °C, respectively), but the 2-seleno-modified ONs (ON2 and ON4) exhibited no improvement in the Tm values (0 and −1 °C, respectively). These observations are in good agreement with previous reports on 2-thiothymidine,17 2′-Omethyl-2-thiouridine,15 2-selenothymidine,20 and 2′,4′BNA/LNA-S2T.18 Notably, the 2-thio and 2-seleno modifications of scpBNA-T and 2′,4′-BNA/LNA-T successfully destabilized the T·G mismatched wobble base pair. Moreover, switching the T·C mismatch to a S2T·C or Se2T·C pair resulted in decrease in Tm by 3–5 °C (ON6 vs. ON1, ON6 vs. ON2, ON7 vs. ON3, and ON7 vs. ON4); thus, the discriminations against T·G and T·C mismatches were greatly improved. However, despite such positive effects of the 2-thio and 2seleno modifications in terms of base recognition, there was no significant enhancement in their overall sequence specificities toward the full complementary ssDNA (difference in Tm between the matched base pair and the most stable mismatched base pair). With the RNA complement (Y = A), ON1–ON4 formed very stable duplexes because of the 2′,4′-bridge structures (Table 3). Among them, 2-thio-modified ON1 and ON3 showed slightly higher Tm values (54 and 55 °C, respectively) that may be attributed at least in part to the strong stacking interactions between S2T and its 3′-nucleobase in the duplex structure.29 In contrast, the 2-seleno-modified ONs (ON2 and ON4) had slightly lower Tm values than the 2-thio-modified counterparts. Because selenium has the poorest ability to form

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Table 2. Tm values of duplexes formed between the modified ONs and ssDNAsa Oligonucleotide sequenceb

Tm (∆Tm = Tm[mismatch] – Tm[match]) (°C)

ON

X

Y=A

Y=G

Y=C

Y=T

T

52

41 (–11)*

37 (–15)

38 (–14)

scpBNA-T

53

43 (–10)*

38 (–15)

40 (–13)

ON5

c

ON6d

2

ON1

scpBNA-S T

54

40 (–14)

34 (–20)

43 (–11)*

ON2

2

scpBNA-Se T

53

37 (–16)

33 (–20)

40 (–13)*

ON7d

2′,4′-BNA/LNA-T

53

42 (–11)*

38 (–15)

41 (–12)

2

ON3

2′,4′-BNA/LNA-S T

55

39 (–16)

35 (–20)

43 (–12)*

ON4

2′,4′-BNA/LNA-Se2T

52

39 (–13)

35 (–17)

41 (–11)*

a

Conditions: 10 mM phosphate buffer (pH 7.2), 100 mM NaCl, and 4 µM each oligonucleotide. The Tm values reflect the averages of at least three measurements. The asterisks indicate the Tm values of the most stable one-base-mismatched duplexes. bThe sequences of the modified ONs and the target ssDNAs are 5′-d(GCGTTXTTTGCT)-3′ and 5′-d(AGCAAAYAACGC)-3′, respectively. cData from reference 9. dData from reference 10. Table 3. Tm values of duplexes formed between the modified ONs and ssRNAsa Oligonucleotide sequenceb

Tm (∆Tm = Tm[mismatch] – Tm[match]) (°C)

ON

X

Y=A

Y=G

Y=C

Y=U

T

48

43 (–5)*

32 (–16)

33 (–15)

scpBNA-T

53

47 (–6)*

35 (–18)

38 (–15)

ON5

c

ON6d

2

ON1

scpBNA-S T

54

42 (–12)*

36 (–18)

42 (–12)*

ON2

scpBNA-Se2T

52

40 (–12)

34 (–18)

41 (–11)*

52

47 (–5)*

36 (–16)

39 (–13)

ON7

d

2′,4′-BNA/LNA-T 2

ON3

2′,4′-BNA/LNA-S T

55

42 (–13)

36 (–19)

44 (–11)*

ON4

2′,4′-BNA/LNA-Se2T

53

40 (–13)

34 (–19)

41 (–12)*

a

Conditions: 10 mM phosphate buffer (pH 7.2), 100 mM NaCl, and 4 µM each oligonucleotide. The Tm values reflect the averages of at least three measurements. The asterisks indicate the Tm values of the most stable one-base-mismatched duplexes. bThe sequences of the modified ONs and the target ssRNAs are 5′-d(GCGTTXTTTGCT)-3′ and 5′-r(AGCAAAYAACGC)-3′, respectively. cData from reference 9. dData from reference 10.

hydrogen bonds, its inclusion might partially disrupt the minor groove hydration involved in the duplex stability.30 Higher Tm values observed for S2T·U base pairs, compared to those of T·U base pairs, can be explained through the unusual sulfurmediated hydrogen bond, as previously reported.31 Again, ON1–ON4 efficiently discriminated the T·G mismatch. Overall mismatch discrimination and hybridization ability toward the full complementary ssRNA were dramatically enhanced by the 2-thio and 2-seleno modifications in conjunction with the bridgings. The differences in Tm of ON1 and ON3 (or ON2 and ON4) were insignificant. CD spectral analysis showed that ON5, ON7, ON3, and ON4 form a B-form duplex with ssDNA and an A-form duplex with ssRNA (Figure S2, Supporting Information). X-ray crystallographic analysis. The X-ray crystal structure of a duplex featuring T·G mismatches has been solved by Hunter and coworkers (PDB ID: 113D, sequence: 5′d(CGCGAATTTGCG)-3′).32,33 In the present study, to investigate the structural basis for the formation of S2T·G base pairs, a self-assembling Hunter’s 2′-deoxydodecamer modified at the fourth position from the 3′-end with 2′,4′-BNA/LNA-S2T (ON8, sequence: 5′-d(CGCGAATTXGCG)-3′, X = 2′,4′BNA/LNA-S2T) was designed and synthesized (see also Table S1, Supporting Information). The crystals were grown in sodium cacodylate buffer (pH 7.0) containing 0.67 mM dodecamer,

Table 4. X-ray data collection and structure refinement statistics 5′-d(CGCGAATTXGCG)-3′a Data collection Space group

P212121

Unit cell parameters, Å

25.94, 41.13, 65.42

Resolution range, Å (last shell) 32.71–1.96 (2.01–1.96) Unique reflections

5,231 (351)

Completeness, %

98.9 (100.0)

Rmerge, %

5.9 (73.7)

I/σ, I

12.1 (2.0)

Redundancy

6.6 (6.6)

Refinement Rwork, %

25.3

Rfree, %

29.7

Bond length, Å

0.010

Bond angle, ° a

2.515 2

X = 2′,4′-BNA/LNA-S T


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Figure 4. X-ray crystal structure of a self-assembling duplex containing S2T(2′,4′-BNA/LNA)·G mismatches (PDB ID: 6ADV) or T·G mismatches (PDB ID: 113D)32: (A) overall structure of the duplex containing S2T(2′,4′-BNA/LNA)·G mismatches (S2T(2′,4′BNA/LNA)·G base pairs are shown with frames), (B) overall structure of the duplex containing T·G mismatches (T·G base pairs are shown with frames), (C) S2T(2′,4′-BNA/LNA)·G mismatched base pair, (D) the reported T·G mismatched base pair, and (E) superimposition of the S2T(2′,4′-BNA/LNA)·G (green) and T·G (yellow) base pairs.

6.7% 2-methyl-2,4-pentanediol, 8 mM spermine, 53 mM KCl, and 8 mM NaCl (8 °C, 28 days), and the structure was successfully solved at a resolution of 1.96 Å (PDB ID: 6ADV). The X-ray crystallographic data collection and structure refinement statistics are summarized in Table 4. The overall helical structure of ON8 (Figure 4A) was quite similar to that of Hunter’s sequence that produced T·G mismatches (Figure 4B). However, the S2T·G base pair had a longer hydrogen-bonding contact between the S(2) of 2thiothymine and the N(1) of guanine (3.3 Å), compared to that of the corresponding T·G base pair (2.7 Å) (Figures 4C–E). Therefore, the improved discriminations observed for the 2′,4′BNA/LNA-S2T- and scpBNA-S2T-modified ONs against T·G mismatches can be explained through weak interactions between the 2-thiothymine and the facing guanine nucleobase. In addition, the S2T·G base pairs were found to have larger opening angles (13.0° and 16.1°) than those of the T·G base pairs (2.3° and 3.0°), presumably because of the large sulfur atoms. It was interesting that the buckle angles of the S2T·G base pairs (23.1° and 24.8°) were also much larger than those of the T·G base pairs (9.9° and 13.4°). These buckle angles (basepair distortions) seem to originate from the 2′,4′-bridge structure in the DNA duplex (C2′-endo puckered B-form duplex). The other local parameters of the flanking base pairs were quite similar (see Tables S2 and S3, Supporting Information). Since this is the first report on the crystal structure of S2T·G mismatched base pairs, the data should be useful for a deeper understanding of the base-pair recognition mechanism of this type of nucleobase and for the design of other artificial nucleobases.

CONCLUSION scpBNA-S2T, scpBNA-Se2T, 2′,4′-BNA/LNA-S2T, and 2′,4′-BNA/LNA-Se2T phosphoramidites were prepared successfully through unified synthetic routes and incorporated into designed ONs. The affinities of the obtained ONs toward full complementary and one-base-mismatched partner strands were evaluated. All the ONs showed strong affinities and high mismatch discrimination capabilities toward the target ssRNA. X-ray crystal studies of a self-assembling sequence featuring 2′,4′-BNA/LNA-S2T revealed that the S2T·G base pair has weak hydrogen-bonding contacts, which presumably translated to the high T·G mismatch discrimination of the 2′,4′BNA/LNA-S2T- and scpBNA-S2T-modified ONs. Attempts to introduce these artificial nucleic acids (scpBNA-S2T, scpBNA-Se2T, 2′,4′-BNA/LNA-S2T, and 2′,4′-BNA/LNASe2T) into therapeutic ONs are ongoing, and the results will be presented in due course.

EXPERIMENTAL SECTION General experimental procedures. Dry acetonitrile, dichloromethane, N,N-dimethylformamide (DMF), ethanol, pyridine, and toluene were used as purchased. 1H, 13C, and 31P nuclear magnetic resonance (NMR) spectra were recorded on a JEOL JNM-AL300 spectrometer. Chemical shift values are expressed in δ values (ppm) relative to internal tetramethylsilane (0.00 ppm), residual CHCl3 (7.26 ppm), or CHD2OD (3.31 ppm) for 1H NMR and chloroform-d1 (77.16 ppm) or methanol-d4 (49.00 ppm) for 13C NMR. For 31P NMR, 5% H3PO4 (0.00 ppm) was used as an external standard. Infra-



red (IR) spectra were recorded on a JASCO FT/IR-4200 spectrometer. Optical rotations were measured on a JASCO DIP370 instrument. Mass spectra of all new compounds were measured on a SpiralTOF JMS-S300 instrument. Mass spectra of ONs were measured on a Bruker Daltonics Autoflex II TOF/TOF mass spectrometer. Flash column chromatography was carried out with Fuji Silysia PSQ-100B, PSQ-60B, or DIOL MB100-40/75. For high-performance liquid chromatography (HPLC), Shimadzu DGU-20A3R, LC-20AD, CBM-20A, CTO-20AC, SPD-20A, and FRC-10A were utilized. For ultraviolet (UV) absorbance measurements, a Shimadzu UV-1800 spectrometer was utilized. Circular dichroism (CD) sprctra were recorded using a JASCO J-720W spectrometer. Synthesis of compound 2. 4-Dimethylaminopyridine (7 mg, 0.06 mmol) and acetic anhydride (30.5 µL, 0.32 mmol) were added to a solution of compound 1 (32 mg, 0.11 mmol) in dry pyridine (1.1 mL) at room temperature under a N2 atmosphere. After the mixture was stirred at room temperature for 40 min, water was added and the mixture was extracted with AcOEt. The organic phase was washed with water, concentrated, and coevaporated with toluene. The residue was purified using flash column chromatography (SiO2, n-hexane:AcOEt = 1:2) to afford 2 (40 mg, quant.) as a white solid. Compound 2: 1H NMR (300 MHz, CDCl3) δ 0.77–1.02 (m, 4H), 1.96 (d, J = 1.4 Hz, 3H), 2.14 (s, 3H), 2.18 (s, 3H), 4.03 (d, J = 12.4 Hz, 1H), 4.26 (d, J = 12.9 Hz, 1H), 4.69 (s, 1H), 4.99 (s, 1H), 5.80 (s, 1H), 7.51 (d, J = 1.4 Hz, 1H), 8.93 (s, 1H); 13C NMR (76 MHz, CDCl3) δ 5.4, 9.7, 13.1, 20.8, 20.9, 58.1, 68.3, 72.5, 78.1, 85.9, 87.1, 110.8, 134.1, 149.8, 163.5, 170.1, 170.1; IR (KBr): 3022, 2840, 1746, 1704, 1239, 1212, 1052 cm−1; [α]D23 +28.4 (c 1.00, CHCl3); HRMS (MALDI) Calcd. for C17H20N2O8Na [M+Na]+: 403.1112, Found: 403.1113. Synthesis of compound 3. Compound 2 (270 mg, 0.71 mmol) was dried using P2O5 under vacuum and dissolved in dry acetonitrile (7.1 mL). 2,4,6-Triisopropylbenzenesulfonyl chloride (279 mg, 0.92 mmol) and potassium carbonate (490 mg, 3.54 mmol) were added at room temperature under a N2 atmosphere. After it was stirred at 60 °C for 2.5 h, the mixture was cooled to room temperature, and then 2,6-dimethylphenol (121 mg, 0.99 mmol) and 1,4-diazabicyclo[2.2.2]octane (24 mg, 0.21 mmol) were added. The resulting solution was stirred at 50 °C for 1 h, and then poured into saturated aq. NH4Cl at 0 °C and extracted with AcOEt. The organic phase was successively washed with water and brine, dried over Na2SO4, and concentrated. The residue was purified using flash column chromatography (SiO2, n-hexane:AcOEt = 1:1) to afford 3 (337 mg, quant.) as a white solid. Compound 3: 1H NMR (300 MHz, CDCl3) δ 0.77–1.01 (m, 4H), 2.11 (s, 3H), 2.13 (s, 3H), 2.15 (s, 3H), 2.21 (s, 6H), 4.03 (d, J = 12.4 Hz, 1H), 4.30 (d, J = 12.9 Hz, 1H), 4.77 (s, 1H), 5.03 (s, 1H), 5.86 (s, 1H), 7.05 (s, 3H), 7.82 (d, J = 0.9 Hz, 1H); 13C NMR (76 MHz, CDCl3) δ 5.3, 9.6, 13.1, 16.6, 16.7, 20.8, 20.9, 58.2, 68.2, 72.3, 77.9, 85.9, 87.9, 104.2, 126.0, 128.8, 139.7, 149.4, 155.2, 170.1, 170.2; IR (KBr): 2927, 1748, 1669, 1537, 1218, 1048 cm−1; [α]D24 +86.2 (c 1.00, CHCl3); HRMS (MALDI) Calcd. for C25H28N2O8Na [M+Na]+: 507.1738, Found: 507.1740. Synthesis of compound 4. Lawesson’s reagent (352 mg, 0.87 mmol) was added to a solution of compound 3 (422 mg, 0.87 mmol) in dry toluene (8.7 mL) at room temperature under a N2 atmosphere. After the mixture was refluxed for 1 h, it was cooled to 0 °C and filtered. The filtrate was concentrated to

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afford 4 (656 mg, crude), which was used for the next reaction without further purification. Synthesis of compound 5. Potassium carbonate (722 mg, 5.23 mmol) and syn-o-nitrobenzaldoxime (434 mg, 2.61 mmol) were added to a solution of compound 4 (656 mg, crude) in dry acetonitrile (8.7 mL) at room temperature under a N2 atmosphere. After it was stirred at 50 °C for 17.5 h, the mixture was cooled to room temperature, and then MeOH (8.7 mL) was added. After this mixture was stirred at 50 °C for 20 min, it was cooled to room temperature and filtered. The filtrate was concentrated, and the residue was purified using flash column chromatography (SiO2, chloroform:MeOH = 12:1) to afford 5 (173 mg, 64%, two steps) as a brown solid. Compound 5: 1H NMR (300 MHz, CD3OD) δ 0.73–0.93 (m, 4H), 1.94 (d, J = 1.4 Hz, 3H), 3.57 (d, J = 12.9 Hz, 1H), 3.74 (d, J = 12.4 Hz, 1H), 4.18 (s, 1H), 4.63 (s, 1H), 6.16 (s, 1H), 7.98 (d, J = 1.4 Hz, 1H); 13C NMR (76 MHz, CD3OD) δ 5.0, 9.9, 12.9, 56.6, 68.6, 71.7, 80.6, 90.6, 91.3, 116.0, 138.2, 163.3, 175.5; IR (KBr): 3076, 2942, 1681, 1496, 1273, 1040 cm−1; [α]D25 −9.5 (c 1.00, MeOH); HRMS (MALDI) Calcd. for C13H16N2O5NaS [M+Na]+: 335.0672, Found: 335.0656. Synthesis of compound 6. 4,4′-Dimethoxytrityl chloride (268 mg, 0.79 mmol) was added to a solution of compound 5 (165 mg, 0.53 mmol) in dry pyridine (10.6 mL) at room temperature under a N2 atmosphere. After the mixture was stirred at room temperature for 4.5 h, water was added, and then was extracted with AcOEt. The organic phase was washed with water, concentrated, and coevaporated with toluene. The residue was purified using flash column chromatography (SiO2, nhexane:AcOEt = 1:1) to afford 6 (311 mg, 96%) as a white solid. Compound 6: 1H NMR (300 MHz, CDCl3) δ 0.51–0.55 (m, 1H), 0.71–0.74 (m, 1H), 0.89–0.98 (m, 2H), 1.69 (d, J = 0.9 Hz, 3H), 2.26 (d, J = 9.2 Hz, 1H), 3.18 (d, J = 11.0 Hz, 1H), 3.34 (d, J = 10.6 Hz, 1H), 3.79 (s, 6H), 4.32 (d, J = 9.6 Hz, 1H), 4.77 (s, 1H), 6.23 (s, 1H), 6.85 (d, J = 8.3 Hz, 4H), 7.24–7.36 (m, 7H), 7.43–7.46 (m, 2H), 7.84 (d, J = 1.4 Hz, 1H), 9.79 (s, 1H); 13C NMR (76 MHz, CDCl3) δ 5.4, 9.7, 12.9, 55.4, 57.7, 67.8, 72.7, 79.2, 87.2, 88.8, 90.1, 113.5, 116.2, 127.3, 128.2, 128.2, 130.2, 130.2, 135.1, 135.3, 136.0, 144.3, 158.9, 160.7, 173.6; IR (KBr): 3432, 2956, 1681, 1508, 1253, 1177, 1046, 833, 766 cm−1; [α]D25 −38.0 (c 1.00, CHCl3); HRMS (MALDI) Calcd. for C34H34N2O7NaS [M+Na]+: 637.1979, Found: 637.1972. Synthesis of compound 7. 2-Cyanoethyl-N,Ndiisopropylphosphoramidochloridite (22.5 µL, 0.10 mmol) was added to a solution of compound 6 (41 mg, 0.07 mmol) and N,N-diisopropylethylamine (34.5 µL, 0.20 mmol) in dry acetonitrile (0.7 mL) at 0 °C under a N2 atmosphere. After it was stirred at room temperature for 4 h, the mixture was concentrated and purified using flash column chromatography (SiO2, n-hexane:AcOEt = 1:1) to afford 7 (43 mg, 78%) as a white solid. Compound 7: 1H NMR (300 MHz, CDCl3) δ 0.38–0.46 (m, 1H), 0.65–0.94 (m, 3H), 1.00 (d, J = 6.5 Hz, 3H), 1.07 (d, J = 6.9 Hz, 3H), 1.12 (d, J = 6.9 Hz, 3H), 1.15 (d, J = 6.5 Hz, 3H), 1.62–1.63 (m, 3H), 2.41 (t, J = 6.2 Hz, 1H), 2.54–2.60 (m, 1H), 3.18–3.30 (m, 2H), 3.46–3.72 (m, 4H), 3.79 (s, 3H), 3.80 (s, 3H), 4.40 (d, J = 6.5 Hz, 0.5H), 4.42 (d, J = 8.3 Hz, 0.5H), 4.95 (s, 0.5H), 4.96 (s, 0.5H), 6.21 (s, 0.5H), 6.22 (s, 0.5H), 6.81–6.88 (m, 4H), 7.21–7.35 (m, 7H), 7.42– 7.46 (m, 2H), 7.89 (d, J = 1.4 Hz, 0.5H), 7.91 (d, J = 1.4 Hz, 0.5H), 9.42 (bs, 1H); 13C NMR (76 MHz, CDCl3) δ 5.5, 5.6, 9.9, 10.0, 13.0, 20.3, 20.5, 20.6, 24.5, 24.6, 24.7, 43.3, 43.4,


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55.3, 55.4, 57.6, 57.7, 57.9, 58.1, 58.3, 68.4, 72.5, 73.1, 77.8, 77.8, 78.7, 78.7, 86.9, 88.7, 88.8, 88.9, 90.8, 90.9, 113.3, 113.4, 116.2, 116.4, 117.4, 117.6, 127.3, 127.4, 128.1, 128.3, 128.3, 130.2, 130.3, 130.4, 135.1, 135.1, 135.2, 135.3, 135.9, 136.0, 144.2, 144.2, 158.8, 158.9, 160.9, 160.9, 173.6, 173.6; 31 P NMR (121.7 MHz, CDCl3) δ 148.7, 149.2; HRMS (MALDI) Calcd. for C43H51N4O8NaPS [M+Na]+: 837.3057, Found: 837.3055. Synthesis of compound 8. 1,8-Diazabicyclo[5.4.0]-7undecene (150 µL, 1.00 mmol) was added to a solution of compound 6 (412 mg, 0.67 mmol) and iodomethane (420 µL, 6.75 mmol) in dry N,N-dimethylformamide (6.7 mL) at 0 °C under a N2 atmosphere. After the mixture was stirred at 0 °C for 1 h, water was added, and then was filtered. The residue was purified using flash column chromatography (SiO2, AcOEt:MeOH = 13:1) to afford 8 (336 mg, 80%) as a pale yellow solid. Compound 8: 1H NMR (300 MHz, CDCl3) δ 0.53–0.63 (m, 1H), 0.77–0.91 (m, 3H), 1.78 (d, J = 0.9 Hz, 3H), 2.61 (s, 3H), 3.09 (d, J = 8.1 Hz, 1H), 3.20 (d, J = 10.8 Hz, 1H), 3.40 (d, J = 11.0 Hz, 1H), 3.78 (s, 6H), 4.38 (d, J = 7.6 Hz, 1H), 4.44 (s, 1H), 5.80 (s, 1H), 6.82–6.85 (m, 4H), 7.21–7.35 (m, 7H), 7.44–7.47 (m, 2H), 7.82 (d, J = 1.2 Hz, 1H); 13C NMR (76 MHz, CDCl3) δ 5.5, 9.8, 14.2, 14.7, 55.4, 57.9, 68.3, 72.2, 80.1, 87.1, 88.3, 113.5, 118.8, 127.3, 128.1, 128.2, 130.1, 130.2, 133.7, 135.1, 135.3, 144.3, 158.8, 160.2, 169.5; IR (KBr): 2933, 1640, 1609, 1509, 1486, 1254, 1177, 1046, 755 cm−1; [α]D21 −25.32 (c 1.00, CHCl3); HRMS (MALDI) Calcd. for C35H36N2O7NaS [M+Na]+: 651.2135, Found: 651.2134. Synthesis of compound 9. Sodium borohydride (62 mg, 1.64 mmol) was added to a solution of selenium (107 mg, 1.36 mmol) in dry ethanol (4.5 mL) at 0 °C under a N2 atmosphere, and the mixture was stirred at the same temperature for 30 min until it became transparent. The solution of generated NaSeH in ethanol was added to compound 8 (261 mg, 0.42 mmol) at 0 °C under a N2 atmosphere. After the mixture was stirred at room temperature for three days, water was added, and then was filtered through Celite. The filtrate was extracted with AcOEt, and the organic phase was successively washed with water and brine, dried over Na2SO4, and concentrated. The residue was purified using flash column chromatography (SiO2, n-hexane:AcOEt = 1:1) to afford 9 (230 mg, 84%) as a pale yellow solid. Compound 9: 1H NMR (300 MHz, CDCl3) δ 0.49–0.57 (m, 1H), 0.68–0.76 (m, 1H), 0.86–1.04 (m, 2H), 1.63 (d, J = 1.0 Hz, 3H), 2.09 (d, J = 10.0 Hz, 1H), 3.19 (d, J = 10.8 Hz, 1H), 3.33 (d, J = 10.8 Hz, 1H), 3.80 (s, 6H), 4.33 (d, J = 8.9 Hz, 1H), 4.88 (s, 1H), 6.30 (s, 1H), 6.84–6.87 (m, 4H), 7.23–7.46 (m, 9H), 7.88 (d, J = 1.2 Hz, 1H), 9.91 (s, 1H); 13C NMR (76 MHz, CDCl3) δ 5.4, 9.7, 13.1, 55.4, 57.6, 67.8, 72.6, 79.6, 87.2, 89.1, 92.2, 113.5, 118.0, 127.4, 128.1, 128.2, 130.2, 130.2, 135.1, 135.2, 136.2, 144.3, 158.9, 160.0, 173.5; IR (KBr): 2936, 1682, 1509, 1255, 1178, 1053, 1038, 757, 606 cm−1; [α]D20 −52.6 (c 1.00, CHCl3); HRMS (MALDI) Calcd. for C34H34N2O7NaSe [M+Na]+: 685.1423, Found: 685.1429. Synthesis of compound 10. N,N-Diisopropylethylamine (380 µL, 2.22 mmol) was added to a solution of compound 9 (98 mg, 0.15 mmol) and 3-iodopropionitrile (200 µL, 2.25 mmol) in dry dichloromethane (1.5 mL) at 0 °C under a N2 atmosphere. After the mixture was stirred at 0 °C for 40 min, additional 3-iodopropionitrile and N,N-diisopropylethylamine were added, and the resulting mixture was stirred until the starting material was consumed. The mixture was quenched with water and extracted with AcOEt. The organic phase was

washed successively with water and brine, dried over Na2SO4, and concentrated. The residue was purified using flash column chromatography (SiO2, AcOEt:MeOH = 12:0, then 12:1) to afford 10 (99 mg, 94%) as a pale yellow solid. Compound 10: 1 H NMR (300 MHz, CDCl3) δ 0.54–0.58 (m, 1H), 0.77–0.96 (m, 3H), 1.78 (d, J = 1.0 Hz, 3H), 2.51 (d, J = 8.9 Hz, 1H), 3.01–3.06 (m, 2H), 3.21 (d, J = 11.0 Hz, 1H), 3.37 (d, J = 11.0 Hz, 1H), 3.42–3.55 (m, 2H), 3.79 (s, 6H), 4.39 (d, J = 8.9 Hz, 1H), 4.42 (s, 1H), 5.69 (s, 1H), 6.82–6.87 (m, 4H), 7.25–7.46 (m, 9H), 7.85 (d, J = 1.2 Hz, 1H); 13C NMR (76 MHz, CDCl3) δ 5.4, 9.8, 14.1, 18.8, 23.8, 55.3, 57.7, 68.4, 71.9, 80.3, 87.1, 88.6, 89.5, 113.4, 118.8, 119.6, 127.2, 128.1, 128.1, 130.1, 130.2, 134.5, 135.0, 135.3, 144.3, 155.0, 158.8, 169.1; IR (KBr): 3006, 2951, 1634, 1609, 1508, 1485, 1253, 1177, 1053, 1039, 834, 752, 581 cm−1; [α]D20 −38.1 (c 1.00, CHCl3); HRMS (MALDI) Calcd. for C37H37N3O7NaSe [M+Na]+: 738.1689, Found: 738.1706. Synthesis of compound 11. 2-Cyanoethyl-N,Ndiisopropylphosphoramidochloridite (65 µL, 0.29 mmol) was added to a solution of compound 10 (139 mg, 0.19 mmol) and N,N-diisopropylethylamine (100 µL, 0.58 mmol) in dry acetonitrile (2.0 mL) at 0 °C under a N2 atmosphere. After it was stirred at room temperature for 2 h, the mixture was concentrated, and the residue was purified using flash column chromatography (DIOL-SiO2, n-hexane:AcOEt = 1:2, then 1:10) to afford 11 (144 mg, 81%) as a white solid. Compound 11: 1H NMR (300 MHz, CDCl3) δ 0.41–0.48 (m, 1H), 0.74–0.92 (m, 3H), 0.99 (d, J = 6.7 Hz, 3H), 1.05 (d, J = 6.9 Hz, 3H), 1.12 (d, J = 6.9 Hz, 3H), 1.15 (d, J = 7.1 Hz, 3H), 1.74 (s, 1.5H), 1.76 (s, 1.5H), 2.40 (t, J = 6.0 Hz, 1H), 2.47–2.67 (m, 1H), 3.02– 3.33 (m, 4H), 3.38–3.69 (m, 6H), 3.79 (s, 3H), 3.80 (s, 3H), 4.45 (d, J = 6.0 Hz, 0.5H), 4.48 (d, J = 7.7 Hz, 0.5H), 4.59 (s, 0.5H), 4.66 (s, 0.5H), 5.69 (s, 0.5H), 5.71 (s, 0.5H), 6.81–6.87 (m, 4H), 7.24–7.35 (m, 7H), 7.42–7.46 (m, 2H), 7.88 (s, 0.5H), 7.92 (s, 0.5H); 13C NMR (76 MHz, CDCl3) δ 5.6, 5.7, 9.9, 10.0, 14.1, 14.2, 18.7, 18.8, 20.3, 20.3, 20.4, 20.5, 23.5, 23.5, 24.4, 24.5, 24.6, 43.2, 43.4, 55.3, 55.3, 57.6, 57.6, 57.7, 57.8, 58.1, 58.3, 68.7, 68.8, 72.0, 72.2, 72.7, 72.9, 78.8, 78.9, 79.6, 79.6, 87.0, 88.4, 88.5, 88.7, 88.7, 89.9, 113.3, 117.4, 117.6, 118.7, 119.0, 119.7, 119.8, 127.2, 127.3, 128.1, 128.2, 130.0, 130.1, 130.2, 133.9, 134.0, 134.9, 134.9, 135.1, 135.2, 144.0, 144.1, 154.5, 154.6, 158.8, 158.8, 168.8, 168.9; 31P NMR (121.7 MHz, CDCl3) δ 149.1, 149.5; HRMS (MALDI) Calcd. for C46H54N5O8NaPSe [M+Na]+: 938.2767, Found: 938.2777. Synthesis of compound 14. Compound 13 (2.33 g, 6.58 mmol) was dried using P2O5 under vacuum and dissolved in dry acetonitrile (66 mL). 2,4,6-Triisopropylbenzenesulfonyl chloride (2.59 g, 8.55 mmol) and potassium carbonate (4.55 g, 32.92 mmol) were added at room temperature under a N2 atmosphere. After the mixture was stirred at 60 °C for 3 h, it was cooled to room temperature, and then 2,6-dimethylphenol (1.13 g, 9.25 mmol) and 1,4-diazabicyclo[2.2.2]octane (221 mg, 1.97 mmol) were added. The resulting solution was stirred at 50 °C for 1 h and then poured into saturated aq. NH4Cl at 0 °C and extracted with AcOEt. The organic phase was washed successively with water and brine, dried over Na2SO4, and concentrated. The residue was purified using flash column chromatography (SiO2, n-hexane:AcOEt = 1:2) to afford 14 (2.88 g, 95%) as a white solid. Compound 14: 1H NMR (300 MHz, CDCl3) δ 2.10 (s, 3H), 2.13 (s, 3H), 2.15 (s, 3H), 2.20 (s, 6H), 3.99 (d, J = 8.3 Hz, 1H), 4.02 (d, J = 8.3 Hz, 1H), 4.40 (d, J = 12.9 Hz, 1H), 4.53 (d, J = 12.9 Hz, 1H), 4.75 (s, 1H), 4.94 (s, 1H), 5.75 (s, 1H), 7.05 (s, 3H), 7.74 (d, J = 0.9 Hz, 1H); 13C



NMR (76 MHz, CDCl3) δ 13.1, 16.6, 16.7, 20.8, 20.9, 59.1, 71.0, 71.9, 77.9, 85.7, 88.2, 104.3, 126.0, 128.9, 139.5, 149.4, 155.1, 169.9, 170.1; IR (KBr): 2956, 1750, 1671, 1534, 1411, 1378, 1222, 1051 cm−1; [α]D22 +111.3 (c 1.00, CHCl3); HRMS (MALDI) Calcd. for C23H27N2O8 [M+H]+: 459.1762, Found: 459.1762. Synthesis of compound 15. Lawesson’s reagent (2.48 g, 6.13 mmol) was added to a solution of compound 14 (2.81 g, 6.13 mmol) in dry toluene (62 mL) at room temperature under a N2 atmosphere. After it was refluxed for 70 min, the mixture was cooled to 0 °C and filtered. The filtrate was concentrated to afford 15 (5.13 g, crude), which was used for the next reaction without further purification. Synthesis of compound 16. Potassium carbonate (5.08 g, 36.8 mmol) and syn-o-nitrobenzaldoxime (3.06 g, 18.4 mmol) were added to a solution of compound 15 (5.13 g, crude) in dry acetonitrile (62 mL) at room temperature under a N2 atmosphere. After it was stirred at 50 °C for 16 h, the mixture was cooled to room temperature, and MeOH (62 mL) was added. The resulting mixture was stirred at 50 °C for 30 min, and then cooled to room temperature and filtered. The filtrate was concentrated, and the residue was purified using flash column chromatography (SiO2, chloroform:MeOH = 8:1) to afford 16 (794 mg, 45%, two steps) as a brown solid. Compound 16: 1H NMR (300 MHz, CD3OD) δ 1.94 (d, J = 1.0 Hz, 3H), 3.75 (d, J = 7.9 Hz, 1H), 3.92 (s, 2H), 3.96 (d, J = 7.6 Hz, 1H), 4.07 (s, 1H), 4.58 (s, 1H), 6.07 (s, 1H) 7.93 (d, J = 1.0 Hz, 1H); 13C NMR (76 MHz, CD3OD) δ 12.9, 57.3, 70.1, 72.2, 80.4, 91.0, 91.5, 116.1, 138.0, 163.3, 175.4; IR (KBr): 3359, 1684, 1493, 1278, 1128, 1054 cm−1; [α]D22 +23.0 (c 1.00, MeOH); HRMS (MALDI) Calcd. for C11H14N2O5NaS [M+Na]+: 309.0516, Found: 309.0512. Synthesis of compound 17. 4,4′-Dimethoxytrityl chloride (1.32 g, 3.90 mmol) was added to a solution of compound 16 (743 mg, 2.59 mmol) in dry pyridine (52 mL) at room temperature under a N2 atmosphere. After the mixture was stirred at room temperature for 3 h, water was added, and then the mixture was extracted with AcOEt. The organic phase was washed with water, concentrated, and coevaporated with toluene. The residue was purified using flash column chromatography (SiO2, n-hexane:AcOEt = 2:3) to afford 17 (1.43 g, 94%) as a yellow solid. Compound 17: 1H NMR (300 MHz, CDCl3) δ 1.65 (s, 3H), 2.49 (d, J = 5.5 Hz, 1H), 3.49 (d, J = 11.0 Hz, 1H), 3.60 (d, J = 11.5 Hz, 1H), 3.79 (s, 6H), 3.81 (d, J = 12.4 Hz, 1H), 3.90 (d, J = 8.3 Hz, 1H), 4.29 (d, J = 4.1 Hz, 1H), 4.76 (s, 1H), 6.11 (s, 1H), 6.85 (m, 4H), 7.24–7.46 (m, 9H), 7.84 (d, J = 0.9 Hz, 1H), 9.88 (s, 1H); 13C NMR (76 MHz, CDCl3) δ 12.9, 55.4, 58.2, 70.6, 71.6, 79.0, 87.0, 89.0, 90.3, 113.5, 113.5, 116.1, 127.4, 128.2, 128.2, 130.2, 130.2, 135.4, 135.4, 136.0, 144.5, 158.9, 160.8, 173.5; IR (KBr): 3415, 1684, 1607, 1508, 1252, 1177, 1073, 1051 cm−1; [α]D22 −24.0 (c 1.00, CHCl3); HRMS (MALDI) Calcd. for C32H32N2O7NaS [M+Na]+: 611.1822, Found: 611.1827. Synthesis of compound 18. 2-Cyanoethyl-N,Ndiisopropylphosphoramidochloridite (60 µL, 0.27 mmol) was added to a solution of compound 17 (104 mg, 0.18 mmol) and N,N-diisopropylethylamine (91 µL, 0.53 mmol) in dry acetonitrile (1.8 mL) at 0 °C under a N2 atmosphere. After it was stirred at room temperature for 3 h, the mixture was concentrated, and the residue was purified using flash column chromatography (SiO2, n-hexane:AcOEt = 3:2) to afford 18 (117 mg, 84%) as a white solid. Compound 18: 1H NMR (300 MHz,

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CDCl3) δ 1.01 (d, J = 6.9 Hz, 3H), 1.10 (d, J = 6.9 Hz, 3H), 1.13 (d, J = 6.9 Hz, 3H), 1.16 (d, J = 6.5 Hz, 3H), 1.55 (d, J = 1.0 Hz, 1.5H), 1.58 (d, J = 1.0 Hz, 1.5H), 2.39 (t, J = 6.2 Hz, 1H), 2.49–2.67 (m, 1H), 3.40–3.72 (m, 6H), 3.79 (s, 3H), 3.80 (s, 3H), 3.83–3.88 (m, 2H), 4.36 (d, J = 6.5 Hz, 0.5H), 4.40 (d, J = 8.3 Hz, 0.5H), 4.89 (s, 0.5H), 4.92 (s, 0.5H), 6.12 (s, 1H), 6.82–6.88 (m, 4H), 7.25–7.35 (m, 7H), 7.42–7.47 (m, 2H), 7.86 (d, J = 1.4 Hz, 0.5H), 7.89 (d, J = 1.4 Hz, 0.5H), 9.39 (bs, 1H); 13C NMR (76 MHz, CDCl3) δ 12.8, 12.8, 20.3, 20.4, 20.5, 20.6, 24.5, 24.6, 24.7, 43.3, 43.5, 55.3, 55.4, 57.8, 58.1, 58.2, 58.3, 58.5, 70.8, 71.0, 71.3, 71.5, 72.1, 72.2, 78.4, 78.4, 86.8, 88.6, 88.7, 88.7, 88.8, 90.8, 90.8, 113.3, 113.4, 116.2, 116.4, 117.4, 117.6, 127.3, 127.4, 128.1, 128.3, 128.4, 130.2, 130.3, 130.3, 130.4, 135.2, 135.2, 135.3, 135.3, 135.8, 135.9, 144.2, 144.3, 158.8, 158.8, 160.9, 160.9, 173.4, 173.5; 31P NMR (121.7 MHz, CDCl3) δ 149.4, 149.6; HRMS (MALDI) Calcd. for C41H49N4O8NaPS [M+Na]+: 811.2901, Found: 811.2899. Synthesis of compound 19. 1,8-Diazabicyclo[5.4.0]-7undecene (160 µL, 1.07 mmol) was added to a solution of compound 17 (414 mg, 0.70 mmol) and iodomethane (440 µL, 7.07 mmol) in dry N,N-dimethylformamide (7.0 mL) at 0 °C under a N2 atmosphere. After the mixture was stirred at 0 °C for 1 h, water was added, and then the mixture was filtered. The residue was purified using flash column chromatography (SiO2, AcOEt:MeOH = 12:1) to afford 19 (338 mg, 80%) as a white solid. Compound 19: 1H NMR (300 MHz, CDCl3) δ 1.74 (s, 3H), 2.61 (s, 3H), 3.36 (d, J = 5.5 Hz, 1H), 3.54 (d, J = 11.0 Hz, 1H), 3.61 (d, J = 11.0 Hz, 1H), 3.79 (s, 6H), 3.82 (d, J = 7.9 Hz, 1H), 3.94 (d, J = 8.3 Hz, 1H), 4.33 (d, J = 5.2 Hz, 1H), 4.41 (s, 1H), 5.69 (s, 1H), 6.83–6.87 (m, 4H), 7.22–7.37 (m, 7H), 7.45–7.48 (m, 2H), 7.82 (s, 1H); 13C NMR (76 MHz, CDCl3) δ 14.1, 14.7, 55.4, 58.4, 70.0, 72.0, 79.8, 86.9, 88.5, 88.6, 113.4, 118.6, 127.3, 128.2, 128.2, 130.2, 130.3, 134.0, 135.4, 135.5, 144.5, 158.8, 160.2, 169.8; IR (KBr): 2951, 1640, 1609, 1509, 1485, 1252, 1177, 1054, 830, 755 cm−1; [α]D23 −11.8 (c 1.00, CHCl3); HRMS (MALDI) Calcd. for C33H34N2O7NaS [M+Na]+: 625.1979, Found: 625.1980. Synthesis of compound 20. Sodium borohydride (116 mg, 3.07 mmol) was added to a solution of selenium (198 mg, 2.51 mmol) in dry ethanol (8.7 mL) at 0 °C under a N2 atmosphere, and the mixture was stirred at the same temperature for 30 min until it became transparent. The solution of generated NaSeH in ethanol was added to compound 19 (487 mg, 0.81 mmol) at 0 °C under a N2 atmosphere. After the mixture was stirred at room temperature for three days, water was added, and then the mixture was filtered through Celite. The filtrate was extracted with AcOEt, and the organic phase was washed successively with water and brine, dried over Na2SO4, and concentrated. The residue was purified using flash column chromatography (SiO2, n-hexane:AcOEt = 1:1) to afford 20 (460 mg, 90%) as a pale yellow solid. Compound 20: 1H NMR (300 MHz, CDCl3) δ 1.60 (d, J = 0.9 Hz, 3H), 2.58 (s, 1H), 3.50 (d, J = 11.2 Hz, 1H), 3.60 (d, J = 11.0 Hz, 1H), 3.79 (s, 6H), 3.82 (d, J = 8.3 Hz, 1H), 3.91 (d, J = 8.3 Hz, 1H), 4.32 (s, 1H), 4.88 (s, 1H), 6.20 (s, 1H), 6.84–6.87 (m, 4H), 7.22–7.36 (m, 7H), 7.44–7.47 (m, 2H), 7.88 (d, J = 1.2 Hz, 1H), 10.38 (s, 1H); 13C NMR (76 MHz, CDCl3) δ 13.1, 55.4, 58.1, 70.5, 71.6, 79.4, 87.1, 89.3, 92.4, 113.5, 113.5, 117.9, 127.4, 128.2, 128.3, 130.2, 135.3, 135.4, 136.1, 144.4, 158.9, 160.1, 173.3; IR (KBr): 2953, 1683, 1508, 1467, 1253, 1178, 1054, 752, 585 cm−1; [α]D23 −39.9 (c 1.00, CHCl3); HRMS (MALDI) Calcd. for C32H32N2O7NaSe [M+Na]+: 659.1270, Found: 659.1255.


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Synthesis of compound 21. N,N-Diisopropylethylamine (1.23 mL, 7.19 mmol) was added to a solution of compound 20 (305 mg, 0.48 mmol) and 3-iodopropionitrile (640 µL, 7.21 mmol) in dry dichloromethane (4.8 mL) at 0 °C under a N2 atmosphere. After the mixture was stirred at 0 °C for 1.5 h, additional 3-iodopropionitrile and N,N-diisopropylethylamine were added, and the mixture was stirred until the starting material was consumed. The mixture was quenched with water and extracted with AcOEt. The organic phase was washed successively with water and brine, dried over Na2SO4, and concentrated. The residue was purified using flash column chromatography (SiO2, AcOEt:MeOH = 12:0, then 12:1) to afford 21 (294 mg, 89%) as a white solid. Compound 21: 1H NMR (300 MHz, CDCl3) δ 1.74 (d, J = 0.9 Hz, 3H), 2.85 (d, J = 5.5 Hz, 1H), 2.93–3.11 (m, 2H), 3.41–3.54 (m, 3H), 3.64 (d, J = 11.2 Hz, 1H), 3.79 (s, 6H), 3.82 (d, J = 8.3 Hz, 1H), 3.92 (d, J = 8.3 Hz, 1H), 4.35 (d, J = 5.7 Hz, 1H), 4.40 (s, 1H), 5.57 (s, 1H), 6.84–6.87 (m, 4H), 7.25–7.37 (m, 7H), 7.45–7.48 (m, 2H), 7.86 (d, J = 1.0 Hz, 1H); 13C NMR (76 MHz, CDCl3) δ 14.1, 18.8, 23.8, 55.4, 58.2, 69.7, 72.0, 80.0, 86.9, 88.7, 89.7, 113.4, 118.8, 119.6, 127.2, 128.2, 128.2, 130.2, 130.3, 134.8, 135.3, 135.4, 144.5, 155.1, 158.8, 169.4; IR (KBr): 2949, 1635, 1609, 1508, 1484, 1251, 1176, 1059 cm−1; [α]D23 −18.8 (c 1.00, CHCl3); HRMS (MALDI) Calcd. for C35H35N3O7NaSe [M+Na]+: 712.1532, Found: 712.1538. Synthesis of compound 22. 2-Cyanoethyl-N,Ndiisopropylphosphoramidochloridite (80 µL, 0.36 mmol) was added to a solution of compound 21 (165 mg, 0.24 mmol) and N,N-diisopropylethylamine (125 µL, 0.73 mmol) in dry acetonitrile (2.4 mL) at 0 °C under a N2 atmosphere. After it was stirred at room temperature for 2 h, the mixture was concentrated, and the residue was purified using flash column chromatography (DIOL-SiO2, n-hexane:AcOEt = 1:2, then 1:10) to afford 22 (175 mg, 82%) as a white solid. Compound 22: 1H NMR (300 MHz, CDCl3) δ 1.00 (d, J = 6.7 Hz, 3H), 1.07 (d, J = 6.7 Hz, 3H), 1.13 (d, J = 6.7 Hz, 3H), 1.16 (d, J = 6.9 Hz, 3H), 1.67 (d, J = 1.0 Hz, 1.7H), 1.71 (d, J = 0.9 Hz, 1.3H), 2.38 (t, J = 6.1 Hz, 1.1H), 2.49–2.68 (m, 0.9H), 3.00–3.12 (m, 2H), 3.43–3.68 (m, 8H), 3.79 (s, 2.6H), 3.80 (s, 3.4H), 3.83– 3.91 (m, 2H), 4.41 (d, J = 6.4 Hz, 0.4H), 4.47 (d, J = 8.3 Hz, 0.6H), 4.54 (s, 0.6H), 4.63 (s, 0.4H), 5.60 (s, 0.4H), 5.61 (s, 0.6H), 6.82–6.88 (m, 4H), 7.25–7.35 (m, 7H), 7.42–7.46 (m, 2H), 7.86 (d, J = 1.0 Hz, 0.4H), 7.90 (d, J = 1.2 Hz, 0.6H); 13C NMR (76 MHz, CDCl3) δ 14.1, 14.1, 18.8, 18.8, 20.3, 20.4, 20.4, 20.5, 23.5, 23.5, 24.5, 24.6, 24.7, 43.4, 43.5, 55.3, 55.3, 57.8, 58.0, 58.0, 58.1, 58.3, 58.6, 70.3, 70.5, 71.0, 71.2, 72.3, 72.4, 78.7, 78.7, 79.3, 79.4, 86.9, 88.4, 88.4, 88.5, 88.6, 89.8, 89.9, 113.3, 113.4, 117.4, 117.6, 118.7, 119.0, 119.8, 119.9, 127.3, 127.4, 128.1, 128.2, 128.3, 130.1, 130.2, 130.3, 130.3, 133.8, 133.8, 135.1, 135.1, 135.3, 144.1, 144.2, 154.4, 154.6, 158.8, 158.8, 168.8, 168.8; 31P NMR (121.7 MHz, CDCl3) δ 149.8; HRMS (MALDI) Calcd. for C44H52N5O8NaPSe [M+Na]+: 912.2611, Found: 912.2610. Synthesis, purification, and characterization of the modified ONs. ONs modified with scpBNA-S2T, scpBNA-Se2T, 2′,4′-BNA/LNA-S2T, or 2′,4′-BNA/LNA-Se2T were synthesized using an nS-8 Oligonucleotide Synthesizer (GeneDesign). The coupling times for these phosphoramidites were extended to 8 min, and 5-[3,5-bis(trifluoromethyl)phenyl]-1H-tetrazole was used as an activator. tert-Butyl hydroperoxide/acetonitrile/water solution (7:60:3) was used for the oxidation (oxidation wait time = 10 min). The other synthetic procedures involved the standard phosphoramidite pro-

tocols. For ONs containing scpBNA-Se2T or 2′,4′-BNA/LNASe2T, columns were initially treated with 0.3 M 1,8diazabicyclo[5.4.0]-7-undecene in acetonitrile (1.0 mL) for 1 h at room temperature to remove the cyanoethyl groups. Cleavage from the solid support and removal of all protecting groups were accomplished by using a 28% ammonia solution (1.5 h at room temperature and then 3 h at 55 °C). In the 4,4′dimethoxytrityl-on (DMTr-on) mode synthesis, the resulting ONs were purified quickly with Sep-Pak® Plus C18 cartridges. In DMTr-off mode synthesis, the resulting ONs were purified quickly with NAPTM-10 SephadexTM G-25 DNA Grade columns. These ONs were further purified using reverse-phase HPLC (Waters XTerra® MS C18 2.5 µm, 10 × 50 mm column, buffer A (0.1 M triethylammonium acetate (TEAA) in water):buffer B (0.1 M TEAA in MeCN) = 1:1, flow rate = 3.0 mL/min). The purified ONs were analyzed using reversephase HPLC (Waters XTerra® MS C18 2.5 µm, 4.6 × 50 mm column), and their compositions were confirmed using matrixassisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) analysis. ON1 was obtained in 35% yield when a 0.02 M iodine solution in pyridine/water/tetrahydrofuran was used as an oxidizer (time for each reaction was 15 s). UV melting experiments. The UV melting experiments were performed on Shimadzu UV-1650B and UV-1800 spectrometers equipped with a Tm analysis accessory. Samples containing an ON (4 µM), the target DNA or RNA (4 µM), and 100 mM NaCl in a 10 mM phosphate buffer (pH 7.2) were annealed at 100 °C and then cooled slowly to room temperature. The melting profile was recorded from 5 to 90 °C at a scan rate of 0.5 °C/min, with detection at 260 nm. The Tm value was obtained from the temperature for half-dissociation of the formed duplexes based on the first derivative of the melting curve. Crystallization. Crystallization conditions were screened with the Nucleic Acid Mini Screen (Hampton Research), using the hanging drop vapor diffusion technique. Droplets consisting of a mixture of ON and the mini-screen buffer were equilibrated against 480 µL of 35% v/v 2-methyl-2,4pentanediol (MPD). A crystal suitable for the diffraction experiment was obtained from a droplet containing 1 µL of 2 mM ON and 2 µL of buffer solution (10% v/v MPD, 40 mM sodium cacodylate trihydrate pH 7.0, 12 mM spermine tetrahydrochloride, 80 mM potassium chloride, and 12 mM sodium chloride). The crystal was mounted in a nylon loop and frozen in liquid nitrogen with the reservoir solution as a cryoprotectant. X-ray data collection and refinement. X-ray diffraction data were collected at SPring-8 (Hyogo, Japan) with a beamline BL44XU equipped with an MX300-HE charge-coupled device (CCD). The initial structure was determined by the molecular replacement method using the single-stranded dodecanucleotide (PDB ID: 1D27)34 as a template model. Rotation and translation searches for the molecular replacement were performed by PHASER.35 The atomic models were built using the molecular graphics program COOT36 and refined using REFMAC.37 A geometric restraint library, used in the refinement of 2′,4′-BNA/LNA-S2T, was produced with CCP4 programs38 Monomer Library Sketcher and Libcheck. Refinement involving non-crystallographic symmetry (NCS) restraints was carried out. The statistics for data collection and



refinement are summarized in Table 4. Base pair parameters were calculated using 3DNA.39 Coordinates. Final coordinates and structure factors have been deposited in the Protein Data Bank, PDB ID code 6ADV (https://www.rcsb.org).

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ASSOCIATED CONTENT

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Supporting Information The Supporting Information is available free of charge on the ACS Publications website. 1 H and 13C NMR spectra for all new compounds, HPLC charts for all new ONs, CD spectra, X-ray crystallography data, PDB file, and supplementary data.

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AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] ORCID Takao Yamaguchi: 0000-0003-3180-0257 Hiroshi Aoyama: 0000-0001-7915-8975 Satoshi Obika: 0000-0002-6842-6812

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Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported in part by JSPS KAKENHI under Grant Number 16K17930, the Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED under Grant Number JP18am0101084, and the Basic Science and Platform Technology Program for Innovative Biological Medicine from AMED under Grant Number JP18am0301004. This work was performed in part using synchrotron beamline BL44XU at SPring-8 under the Cooperative Research Program of the Institute for Protein Research, Osaka University. Diffraction data were collected at Osaka University beamline BL44XU at SPring-8 (Harima, Japan) under proposal number 2014A6902.

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REFERENCES 1

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Obika, S.; Nanbu, D.; Hari, Y.; Morio, K.; In, Y.; Ishida, T.; Imanishi, T. Synthesis of 2′-O,4′-C-methyleneuridine and -cytidine. Novel bicyclic nucleosides having a fixed C3′-endo sugar puckering. Tetrahedron Lett. 1997, 38, 8735–8738. Obika, S.; Nanbu, D.; Hari, Y.; Andoh, J.; Morio, K.; Doi, T.; Imanishi, T. Stability and structural features of the duplexes containing nucleoside analogues with a fixed Ntype conformation, 2′-O,4′-C-methyleneribonucleosides. Tetrahedron Lett. 1998, 39, 5401–5404. Singh, S. K.; Nielsen, P.; Koshkin, A. A.; Wengel, J. LNA (locked nucleic acids): synthesis and high-affinity nucleic acid recognition. Chem. Commun. 1998, 455–456.

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