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Design and Synthesis of Triazole-Linked xylo-Nucleoside Dimers a

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Smriti Srivastava , Sunil K. Singh , Vivek K. Sharma , Priyanka a

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Mangla , Carl E. Olsen & Ashok K. Prasad

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Bioorganic Laboratory, Department of Chemistry, University of Delhi, Delhi, India b

Department of Chemistry, Kirorimal College, University of Delhi, Delhi, India c

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Faculty of Life Sciences, Department of Plant and Environmental Sciences, University of Copenhagen, Copenhagen, Denmark Published online: 12 May 2015.

To cite this article: Smriti Srivastava, Sunil K. Singh, Vivek K. Sharma, Priyanka Mangla, Carl E. Olsen & Ashok K. Prasad (2015) Design and Synthesis of Triazole-Linked xylo-Nucleoside Dimers, Nucleosides, Nucleotides and Nucleic Acids, 34:6, 388-399, DOI: 10.1080/15257770.2015.1004341 To link to this article: http://dx.doi.org/10.1080/15257770.2015.1004341

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Nucleosides, Nucleotides and Nucleic Acids, 34:388–399, 2015 C Taylor and Francis Group, LLC Copyright  ISSN: 1525-7770 print / 1532-2335 online DOI: 10.1080/15257770.2015.1004341

DESIGN AND SYNTHESIS OF TRIAZOLE-LINKED XYLO-NUCLEOSIDE DIMERS

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Smriti Srivastava,1 Sunil K. Singh,1,2 Vivek K. Sharma,1 Priyanka Mangla,1 Carl E. Olsen,3 and Ashok K. Prasad1 1 Bioorganic Laboratory, Department of Chemistry, University of Delhi, Delhi, India 2 Department of Chemistry, Kirorimal College, University of Delhi, Delhi, India 3 Faculty of Life Sciences, Department of Plant and Environmental Sciences, University of Copenhagen, Copenhagen, Denmark Three triazole-linked nonionic xylo-nucleoside dimers T L-t-T xL, T L-t-A BzxL and T L-t-C BzxL have been synthesized for the first time by Cu(I) catalyzed azide-alkyne [3 + 2] cycloaddition reaction (CuAAC) of 1-(3 -azido-3 -deoxy-2 -O,4 -C-methylene-β-D-ribo-furanosyl)thymine with different alkynes, i.e., 1-(5 -deoxy-5 -C-ethynyl-2 -O,4 -C-methylene-β-D-xylofuranosyl)thymine, 9-(5 deoxy-5 -C-ethynyl-2 -O,4 -C-methylene-β-D-xylo-furanosyl)-N6-benzoyladenine and 1-(5 -deoxy-5 C-ethynyl-2 -O,4 -C-methylene-β-D-xylofuranosyl)-N4-benzoylcytosine in 90%–92% yields. Among the two Cu(I) reagents, CuSO4 .5H2 O-sodium ascorbate in THF:tBuOH:H2 O (1:1:1) and CuBr.SMe2 in THF used for cycloaddition (click) reaction, the former one was found to be better yielding than the latter one. 2

Keywords Click chemistry; Huisgen-Sharpless-Meldal [3+2] cycloaddition; locked nucleic acid; phosphate backbone modification

INTRODUCTION Inhibition of gene expression by antisense oligonucleotides has gained much attention as a promising drug design concept, since its inception in 1978.[1–3] Successful drug development based on this technique requires the synthesis and use of chemically modified oligonucleotides that render stability to nucleolytic digestion, enhance cellular uptake, hybridise with high affinity, and specificity toward the targeted mRNA/DNA.[4] Among the sugar-modified nucleosides, 2 -O,4 -C methylene bridge containing oligonucleotides such as β-D-ribo-LNA and β-D-xylo-LNA hybridize to both DNA Received 11 November 2014; accepted 2 January 2015. Address correspondence to Professor Ashok K. Prasad, PhD, University of Delhi, Bioorganic Laboratory, New Delhi 110007, India. E-mail: [email protected] Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lncn.

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FIGURE 1 Structure of DNA / RNA (1a); LNA (1b); triazole-linked 10 mer (1c).

and RNA with high affinity. The experiments toward slightly mismatched RNA targets showed that hybridization of β-D-xylo-configured LNA toward RNA is selective.[5] Recent efforts have been expanded to the development of oligonucleotide analogs with modified phosphate backbone structure, to circumvent the physical and biological limitations of natural phosphodiester linkage.[6–9] The possible benefits of innovative and new synthetic approaches, such as the Cu(I)-catalysed azide-alkyne cycloaddition reaction for the synthesis of modified nucleosides, nucleoside bioconjugates, and oligonucleotides have drawn the attention of the researchers.[10] Lucas, et al.[11] has synthesized the triazole-linked 3 -5 -thymidine dimer and pentamer by Cu-catalysed [3+2] cycloaddition of azido and propargyl precursors. Isobe, et al.[12] has synthesized the triazole linked 10-mer (1c, Figure 1) analog of thymine DNA which leads to much higher melting temperature (T m = 61.1◦ C) compared to natural DNA d(T)10 (T m = 20.0◦ C).[12,13] In continuation of our recent report on synthesis of triazole-linked bicyclo ribonucleoside dimers,[14] we herein report for the first time the design and synthesis of triazole-linked bicyclic xylo-nucleoside dimers 2–4 comprising of preorganized sugar moiety (Figure 2).

RESULTS AND DISCUSSION It was envisaged to synthesize triazole-linked bicyclic xylo-nucleoside dimers, i.e. 1-(3 -deoxy-2 -O,4 -C-methylenethymidin-3 -yl)-4-(1 -(5-deoxy2-O,4-C-methylene-β-D-xylo-furanosyl)thymin-5-yl)-1,2,3-triazole (2), 1-(3 deoxy-2 -O,4 -C-methylenethymidin-3 -yl)-4-(9-(5-deoxy-2-O,4-C-methyle ne-β-D-xylofuranosyl)-N 6-benzoyladenin-5-yl)-1,2,3-triazole (3), 1-(3 -deo xy-2 -O,4 -C-methylenethymidin-3 -yl)-4-(1 -(5-deoxy-2-O,4-C-methyleneβ-D-xylofuranosyl)-N 4-benzoylcytosin-5-yl)-1,2,3-triazole (4) by [3+2] cycl oaddition reaction[15] of 1-(3 -azido-3 -deoxy-2 -O,4 -C-methylene-β-D-ribofu

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FIGURE 2 Structures of triazole-linked bicyclic xylo-nucleoside dimers. The natural phosphate linkage, O-P-O, has been replaced by triazole linkage, N-C-C, shown in red.

ranosyl)thymine (5)[16] with different alkynes, i.e. 1-(5 -deoxy-5 -C-ethynyl2 -O,4 -C-methylene-β-D-xylofuranosyl)thymine (6), 9-(5 -deoxy-5 -C-ethyn yl-2 -O,4 -C-methylene-β-D-xylofuranosyl)-N 6-benzoyladenine (7), and 1-(5 deoxy-5 -C-ethynyl-2 -O,4 -C-methylene-β-D-xylofuranosyl)-N 4-benzoylcytosine (8), respectively (Scheme 3). The synthesis of azido compound 5 and alkynes 6–8 can be accomplished from a common synthon 4-Chydroxymethyl-1,2-O-isopropylidene-β-L-threo-pentofuranose (9), which can easily be obtained from D-glucose following the literature procedure as described by Youssefyeh, et al.[17]

SCHEME 1 Retro-synthetic analysis of triazole-linked nucleoside dimers 2–4.

The synthesis of 1-(3 -azido-3 -deoxy-2 -O,4 -C-methylene-β-D-ribofuranosyl)thymine (5) was efficiently accomplished from furanose triol 9 in an overall yields of 13% by following our previously reported procedure.[16] The alkynylated nucleosides 6–8 were synthesized starting with the same

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furanose-triol 9 in an overall yields of 15%, 16%, and 18%, respectively (Scheme 2). The furanose triol 9 was converted to 5-deoxy-5-C-ethynyl-1,2O-isopropylidene-4-C-methanesulfonyloxymethyl-β-D-xylo-furanose (10) following oxetane chemistry in an overall yield of 34%.[14] The free –OH group at C-3 position in compound 10 was acetylated using acetic anhydride in dichloro-methane:pyridine (1:1) to yield compound 11 in 95% yield, which on acetolysis with acetic acid-acetic anhydride-conc. sulfuric acid (100:10:0.1) provided an anomeric mixture of triacetate 12a–12b in 82% yield. The ¨ Vorbruggen coupling[18] of 12a–12b with thymine, N6-benzoyladenine and cytosine yielded nucleosides 13–15 in 60, 75, and 85% yields, respectively. Treatment of nucleosides 13–15 with 2M NaOH in water:dioxane (1:1) led to the deacetylation followed by cyclisation to afford the bicyclic nucleosides 6, 7, and 16 in 95%, 80%, and 90% yields, respectively. The benzoylation of exocyclic amino group in cytosine nucleoside 16 with benzoic anhydride in DMF afforded 1-(5 -deoxy-5 -C-ethynyl-2 -O,4 -C-methylene-β-D-xylofuranosyl)-N 4benzoylcytosine (8) in 90% yield (Scheme 2).

SCHEME 2 Synthesis of 5 -C-ethynyl bicyclic xylo-nucleosides 6–8.

The three triazole-linked bicyclic xylo-nucleoside dimers 2–4 were synthesized by Cu(I)-catalyzed ‘click’ reaction between bicyclic azidonucleosides 5 with alkynylated bicyclic nucleosides 6, 7, and 8, respectively. Use of two Cu(I) coupling reagents, viz. CuSO4 .5H2 O-sodium ascorbate in THF:tBuOH:H2 O (1:1:1) and CuBr.SMe2 in THF resulted in the formation of triazole-linked dimer 2 in 92% and 75%, dimer 3 in 90% and 65% and

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dimer 4 in 92% and 70% yields, respectively (Scheme 3). The yields are better in case of CuSO4 .5H2 O-sodium ascorbate than in CuBr.SMe2 because of the in situ formation of active Cu(I) catalyst. The direct use of Cu(I) source, such as CuBr.SMe2 results in the formation of side products.[19]

SCHEME 3 Synthesis of triazole-linked bicyclic nonionic xylo-nucleoside dimers TL-t-TxL 2, TL-t-ABzxL 3 and TL-t-CBzxL 4.

The structures of all the synthesized compounds 2–16 were unambiguously established on the basis of their spectral (IR, 1H-, 13C NMR, and HRMS) data analysis. The structures of known compounds 5[16], 9[17], and 10[14] were further confirmed by the comparison of their physical and spectral data with those reported in the literature. CONCLUSION In summary, Copper(I)-catalyzed Huisgen-Sharpless-Meldal [3+2] cycloaddition ‘click’ reaction between azido- and alkynyl-nucleosides has been explored for the synthesis of triazole-linked nonionic bicyclic xylo-nucleoside dimers, i.e. TL-t-TxL, TL-t-ABzxL, and TL-t-CBzxL. Among the used Cu(I) catalyst for click reaction, CuSO4 .5H2 O-sodium ascorbate found to be the better yielding than CuBr.SMe2 in THF. The successful inclusion of structural diversity such as preorganized xylo sugar moiety and neutral internucleoside linkage renders the researchers to scrutinize chemically modified oligonucleotides for wider range of biological applications. EXPERIMENTAL SECTION General Reactions were conducted under an atmosphere of nitrogen, when anhydrous solvents were used. Column chromatography was carried out using

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silica gel (100–200 mesh). Melting points were determined on Buchi-M 560 instrument and are uncorrected. Analytical TLCs were performed on precoated Merck silica gel 60F254 plates; the spots were detected either using UV light or by charring with 4% alcoholic sulfuric acid. The IR spectra were recorded on a Perkin-Elmer model 2000 FT-IR spectrometer by making KBr disc for solid samples and thin films for oils. The optical rotations were measured with Rudolph autopol II automatic polarimeter using light of 546 nm wavelength. The 1H and 13C NMR spectra were recorded on a Bruker Avance AC-300 spectrometer/JEOL alfa-400 spectrometer at 300/400 and 75.5/100.6 MHz, respectively. The chemical shift values are reported as δ ppm relative to TMS used as internal standard and the coupling constants (J ) are measured in Hz. Mass spectra were recorded on JEOL JMSAX505W/Agilent-G6530AA high-resolution mass spectrometer in positive ion mode. Chemicals were obtained from commercial suppliers and were used without any further purification unless otherwise noted. THF, chloroform, methanol, petroleum ether, and ethyl acetate were distilled over Na wire, CaCl2 , CaO, P2 O5 , and K2 CO3 , respectively. 3-O-Acetyl-5-deoxy-5-C-ethynyl-1,2-O-isopropylidene-4-C-methanesulfonyloxymethyl-α-D-xylofuranose (11). To a solution of compound 10 (7.32 g, 23.89 mmol) in dry dichloromethane:pyridine (1:1) was added acetic anhydride (2.8 mL, 29.62 mmol) and the reaction was kept on stirring. After completion of the reaction as shown on analytical TLC, it was quenched by addition of ice cold water, and resulting mixture was extracted with chloroform. Organic layer was washed with bicarbonate, brine and water, and was dried over sodium sulfate. The solvent was removed in vacuo and the product thus obtained was purified by silica gel column chromatography using methanol in chloroform as gradient solvent system to afford compound 11 (7.90 g, 95%) as white solid. Rf = 0.5 (30% ethyl acetate in petroleum ether). M. Pt.: 86–88◦ C; [α]D 35 = −21.4 (c 0.1, CHCl3 ); IR (KBr)νmax: 2989, 2942, 2122, 1752, 1459, 1427, 1359, 1227, 1175, 1063, 1010, 966, 848, and 755 cm−1; 1H NMR (CDCl3 , 300 MHz): δ 5.98 (1H, d, J = 3.9 Hz), 5.26 (1H, s), 4.63 (1H, d, J = 10.8 Hz), 4.59 (1H, d, J = 4.2 Hz), 4.49 (1H, d, J = 10.8 Hz), 3.13 (3H, s), 2.73 (1H, dd, J = 16.8 and 2.4 Hz), 2.55 (1H, d, J = 16.8 and 2.4 Hz), 2.15 (3H, s), 2.02 (1H, t, J = 2.7 Hz) 1.63 (3H, s) and 1.30 (3H, s,); 13C NMR (CDCl3 , 75.5 MHz): δ 169.2, 112.9, 105.6, 87.1, 84.9, 78.1, 76.3, 71.3, 69.0, 37.9, 26.0, 25.5, 22.3 and 20.6. HRMS: m/z calculated for [C14 H20 O8 S+Na+] 371.0771, observed 371. 0763. 1,2,3-Tri-O-acetyl-5-deoxy-5-C-ethynyl-4-C-methanesulfonyloxy-methyl-α, β-D-xylofuranose (12a–12b). Acetic anhydride (21.8 mL, 230.62 mmol) and concentrated sulfuric acid (0.2 mL, 3.75 mmol) was added to a stirred solution of compound 11 (7.2 g, 20.67 mmol) in acetic acid (131.6 mL, 23.01 mmol) and the stirring was continued for 5 h at room temperature. Then, the mixture was quenched by the addition of water (100 mL) and

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stirred for 30 min at room temperature. The mixture was washed with water, bicarbonate solution and brine, and then dried over sodium sulfate. After removal of excess of solvent under vacuo, the residue was purified by silica gel column chromatography using methanol in chloroform as gradient solvent system to give an anomeric mixture (α:β = ca. ∼ 1:1) of 12a–12b (6.65 g, 82%) as colorless viscous oil. Rf = 0.5 (30% ethyl acetate in petroleum ether); IR (KBr)νmax: 3027, 2943, 2123, 1752, 1429, 1362, 1218, 1177, 1057, 1010, 967, 903, 833, and 756 cm−1; 1H NMR (CDCl3 , 300 MHz): δ 6.38 (d, J = 4.2 Hz), 6.16 (s), 5.49–5.25 (m), 4.54–4.44 (m), 3.10–3.09 (s), 2.74–2.47 (m), 2.16–2.07 (m); 13C NMR (CDCl3 , 75.5 MHz): δ 170.3, 169.7, 169.5, 169.3, 168.9, 98.8, 91.5, 86.3, 82.1, 80.9, 77.8, 77.6, 75.8, 74.1, 73.9, 71.7, 69.7, 37.8, 37.6, 23.4, 21.0, 20.9, 20.7, 20.6, 20.3; HRMS: m/z calculated for [C15 H20 O10 S+Na+] 415.0669, observed 415.0671. General procedure for the synthesis of nucleosides 13, 14, and 15. N ,Obis(trimethysilyl)acetamide (BSA) (12.5 mL, 50.29 mmol) was added to an anomeric mixture of sugar derivatives 12a–12b (5.60 g, 14.27 mmol) and thymine/cytosine/N 6-benzyoladenine (18.86 mmol) in dry acetonitrile (120 mL), under nitrogen atmosphere. Dichloroethane was used as solvent for coupling of N 6-benzoyladenine instead of acetonitrile. The reaction mixture was refluxed for 1 h to get a clear solution, brought to RT and then trimethysilyl triflate (TMSOTf) (4.0 mL, 22.10 mmol) was added. The reaction mixture was refluxed for 10–14 h, cooled to room temperature and was extracted with chloroform (3 × 100 mL). The organic layer was washed with sat. NaHCO3 solution (3 × 100 mL) and brine (2 × 50 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and residue thus obtained was purified by silica gel column chromatography using methanol in chloroform as a gradient solvent. 1-(2 ,3 -Di-O-acetyl-5 -deoxy-5 -C-ethynyl-4 -C-methanesulfonyloxy-methyl-β-D-xylofuranosyl)thymine (13). It was obtained as white foam solid (3.92 g, 60%). Rf = 0.5 (10% methanol in chloroform). M. Pt.: 67–69◦ C; [α]D 35 = −51.1 (c 0.1, CHCl3 ); IR (KBr)νmax: 3278, 3025, 2123, 1751, 1694, 1464, 1362, 1226, 1176, 1052, 1002, 967, 831, and 756 cm−1; 1H NMR (CDCl3 , 300 MHz): δ 8.69 (1H, brs, deuterium exchangeable NH), 7.38 (1H, s), 6.09 (1H, d, J = 6.3 Hz), 5.60 (1H, d, J = 5.7 Hz), 5.53 (1H, d, J = 5.4 Hz), 4.44 (2H, d, J = 10.8 Hz), 3.14 (3H, s), 2.74 (1H, dd, J = 17.1 & 2.7 Hz), 2.62 (1H, dd, J = 17.1 and 2.7 Hz), 2.18 (3H, s), 2.12 (3H, s), 1.95 (3H, s), 1.66 (1H, s); 13C NMR (CDCl3 , 75.5 MHz): δ 169.8, 169.6, 163.1, 150.1, 135.1, 112.0, 86.1, 83.0, 77.9, 75.1, 72.4, 69.9, 67.9, 37.8, 23.2, 20.6, 20.5, 12.6; HRMS: m/z calculated for [C18 H22 N2 O10 S+Na+] 481.0887, observed 481.0875. 9-(2 ,3 -Di-O-acetyl-5 -deoxy-5 -C-ethynyl-4 -C-methanesulfonyloxy-methyl-β-D-xylofuranosyl)-N 6-benzoyladenine (14). It was obtained as white foam solid (6.12 g, 75%). M. Pt.: 94–95◦ C; [α]D 30 = −20.85 (c 0.1, MeOH); IR (KBr) νmax: 3284, 2917, 2849, 1752, 1604, 1459, 1363, 1225, 1176, 1054,

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1001, 967, 823, and 712 cm−1; 1H NMR (CDCl3 , 400 MHz): δ 9.11 (1H, brs), 8.82 (1H, s), 8.26 (1H, s), 8.02 (2H, d, J = 8.0 Hz), 7.60 (1H, t, J = 7.6 Hz), 7.51 (2H, t, J = 8.0 Hz), 6.36 (1H, t, J = 6.0 Hz), 6.25 (1H, d, J = 6.0 Hz), 5.71 (1H, d, J = 6.0 Hz), 4.53 (2H, q, J = 11.2 Hz), 3.17 (3H, s), 2.95 (1H, dd, J = 16.8 & 2.8 Hz), 2.79 (1H, dd, J = 17.6 & 2.8 Hz), 2.19 (3H, s), 2.12 (1H, t), 2.09 (3H, s); 13C NMR (CDCl3 , 100.6 MHz): δ 171.5, 166.8, 154.5, 153.9, 151.7, 143.9, 135.3, 135.1, 132.3, 131.0, 130.1, 129.1, 125.3, 87.8, 85.9, 79.7, 79.4, 77.0, 74.4, 72.2, 40.0, 25.6, 22.8, 22.6; HRMS: m/z calculated for [C25 H25 N5 O9 S+H]+ 572.1446, observed 572.1452. 1-(2 ,3 -Di-O-acetyl-5 -deoxy-5 -C-ethynyl-4 -C-methanesulfonyloxy-methyl-β-D-xylofuranosyl)cytosine (15). It was obtained as white solid (5.38 g, 85%). M. Pt.: 145–147◦ C; [α]D 30 = −10.84 (c 0.1, MeOH); IR (KBr) νmax: 3293, 1751, 1648, 1492, 1363, 1225, 1175, 1051, 967, 831, and 755 cm−1; 1H NMR (CDCl3 , 400 MHz): δ 7.95 (1H, brs), 7.49 (1H, d, J = 7.2 Hz), 6.44 (1H, brs), 6.13 (1H, d, J = 6.0 Hz), 5.87 (1H, d, J = 7.6 Hz), 5.51 (1H, d, J = 6.0 Hz), 5.42 (1H, d, J = 5.2 Hz), 4.40 (2H, q, J = 12.4 Hz), 3.07 (3H, s), 2.60 (2H, q, J = 2.4 Hz), 2.11 (1H, t, J = 2.4 Hz), 2.08 (3H, s), 2.03 (3H, s); 13 C NMR (CDCl3 , 100.6 MHz): δ 171.5, 167.7, 157.5, 142.2, 98.1, 88.7, 85.0, 79.7, 79.4, 79.0, 77.0, 74.1, 71.7, 39.5, 31.4, 24.9, 22.5; HRMS: m/z calculated for [C17 H21 N3 O9 S+H]+ 444.1071, observed 444.1065. General procedure for the synthesis of nucleosides 6, 7, and 16. To a stirred solution of compound 13–15 (9.19 mmol) in water:dioxane (22 mL, 1:1) was added 2M NaOH (21.4 mL) and reaction mixture was stirred at RT for 1 h. The reaction mixture was extracted with ethyl acetate (3 × 100 mL), washed with bicarbonate (2 × 100 mL) and brine solution (2×100 mL), dried over sodium sulfate, and concentrated under reduced pressure. The residue thus obtained was purified by silica gel column chromatography using methanol in chloroform as gradient solvent system. 1-(5 -Deoxy-5 -C-ethynyl-2 -O,4 -C-methylene-β-D-xylofuranosyl)-thymine (6). It was obtained as light yellow solid (2.42 g, 95%). Rf = 0.4 (10% methanol in chloroform). M. Pt.: decomposes at 166–168◦ C. [α]D 35 = +5.1 (c 0.1, MeOH); IR (KBr) νmax: 3302, 3223, 3023, 2118, 1689, 1694, 1481, 1417, 1312, 1275, 1143, 1113, 1059, 1030, 996, 901, and 705 cm−1; 1H NMR (DMSO-d 6 , 300 MHz): δ 11.25 (1H, brs), 7.58 (1H, s), 5.83 (1H, d, J = 2.7 Hz), 5.48 (1H, s), 4.22 (1H, s), 4.02 (1H, s), 3.95 (1H, d, J = 10.8 Hz), 3.75 (1H, d, J = 10.8 Hz), 2.96 (1H, s), 2.81 (2H, s), 1.73 (3H, s); 13C NMR (DMSO-d 6 , 75.5 MHz): δ 164.0, 150.1, 137.1, 106.1, 88.3, 87.1, 79.0, 77.6, 73.3, 73.1, 72.6, 16.4, 12.3; HRMS: m/z calculated for [C13 H14 N2 O5 +H+] 279.0975, observed 279.0966. 9-(5 -Deoxy-5 -C-ethynyl-2 -O,4 -C-methylene-β-D-xylofuranosyl)-N 6benzoyladenine (7). It was obtained as white solid (2.84 g, 80%). M. Pt.: 105–107◦ C; [α]D 30 = −19.17 (c 0.1, MeOH); IR (KBr) νmax: 3285, 2923, 2852, 1701, 1611, 1508, 1334, 1253, 1214, 1178, 1066, 1029, 989, 848, 711, and 641 cm−1; 1H NMR (CDCl3 , 400 MHz): δ 9.28 (1H, brs), 8.62 (1H, s),

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8.44 (1H, s), 7.96 (2H, d, J = 8 Hz), 7.56 (1H, t, J = 7.2 Hz), 7.47 (2H, t, J = 7.2 Hz), 6.22 (1H, t, J = 4.4 Hz), 5.43 (1H, brs), 4.65 (1H, d, J = 6.4 Hz), 4.42 (1H, d, J = 2.0 Hz), 4.10 (1H, t, J = 4.4 Hz), 3.97 (1H, t, J = 4.4 Hz), 2.83 (2H, s), 2.04 (1H, s); 13C NMR (CDCl3 , 100.6 MHz): δ 165.0, 152.1, 151.3, 148.8, 143.4, 133.2, 132.8, 128.7, 127.9, 122.4, 87.5, 87.1, 78.8, 76.6, 74.0, 73.5, 71.2, 16.8; HRMS: m/z calculated for [C20 H17 N5 O4 +H]+ 392.1353, observed 392.1349. 1-(5 -Deoxy-5 -C-ethynyl-2 -O,4 -C-methylene-β-D-xylofuranosyl)-cytosine (16). It was obtained as white solid (1.27 g, 90%). M. Pt.: 192–194◦ C; [α]D 30 = +32.56 (c 0.1, MeOH); IR (KBr) νmax: 3489, 3336, 2959, 2887, 1647, 1483, 1273, 1094, 902, 787, 631, and 599 cm−1; 1H NMR (CDCl3 , 400 MHz): δ 7.69 (1H, d, J = 7.2 Hz), 7.03 (2H, brs), 5.77 (1H, brs), 5.62 (1H, d, J = 7.2 Hz), 5.49 (1H, s), 4.20 (1H, d, J = 2.4 Hz), 4.00 (1H, d, J = 1.2 Hz), 3.98 (1H, d, J = 8.0 Hz), 3.76 (1H, d, J = 8.0 Hz), 2.97 (1H, t, J = 2.4 Hz), 2.79 (2H, dd, J = 8.0 and 2.4 Hz); 13C NMR (CDCl3 , 100.6 MHz): δ 165.9, 155.0, 142.1, 91.6, 89.1, 86.7, 86.4, 79.1, 73.2, 72.7, 62.8, 16.5; HRMS: m/z calculated for [C12 H13 N3 O4 +H]+ 264.0906, observed 264.0972. 1-(5 -Deoxy-5 -C-ethynyl-2 -O,4 -C-methylene-β-D-xylofuranosyl)-N 4benzoylcytosine (8). To a solution of compound 16 (0.5 g, 1.90 mmol) in anhydrous DMF (10 mL) was added benzoic anhydride (0.37 g, 3.02 mmol). The mixture was stirred at 25 ◦ C for 6 h, and upon completion of the reaction on analytical TLC, the excess of the solvent was removed under reduced pressure. The crude product thus obtained was purified by silica gel column chromatography as white solid (0.62 g, 90%). M. Pt.: 130–132◦ C; [α]D 30 = + 30.80 (c 0.1, MeOH); IR (KBr) νmax: 3431, 1640, 1485, 1262, 1086, 1048, 791, and 685 cm−1; 1H NMR (CDCl3 , 400 MHz): δ 9.15 (1H, brs), 8.23 (1H, d, J = 6.8 Hz), 7.81 (2H, d, J = 8 Hz), 7.57–7.27 (4H, m), 5.69 (1H, s), 5.69 (1H, brs), 4.82 (1H, s), 4.33 (1H, s), 4.13 (1H, d, J = 8.8 Hz), 3.96 (1H, d, J = 8.8 Hz), 2.90 (2H, s), 2.13 (1H, d, J = 2.8 Hz); 13C NMR (CDCl3 , 100.6 MHz): δ 166.5, 162.4, 155.3, 146.7, 132.9, 132.8, 128.6, 127.7, 95.3, 90.2, 88.1, 77.9, 77.7, 73.8, 73.5, 71.2, 17.1; HRMS: m/z calculated for [C19 H17 N3 O5 +H]+ 368.1241, observed 368. 1227. General Procedure for Huisgen-Sharpless-Meldal [3+2] Cycloaddition Reaction of Compounds 5 with 6–8: Synthesis of Nucleoside Dimers 2, 3, and 4 Method A: Azidonucleoside 5 (0.43 g, 1.45 mmol) and 5 -alkynylated nucleoside 6–8 (1.25 mmol) were suspended in a mixture of THF:tBuOH:H2 O (1:1:1) (60 mL). Sodium ascorbate (0.10 g, 0.50 mmol) was added into the reaction mixture followed by the addition of CuSO4 .5H2 O (0.063 g, 0.25 mmol). The heterogeneous mixture was stirred vigorously for 12 h. After completion of reaction as shown on analytical TLC, excess of solvents

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were evaporated and traces of moisture was removed by coevaporation with ethanol. The crude thus obtained was purified by using silica gel column chromatography. Method B. A mixture of azidonucleoside 5 (0.43 g, 1.45 mmol), 5 alkynylated nucleoside 6–8 (1.25 mmol), and copper bromide dimethyl sulfide complex (CuBr.SMe2 ) (0.40 g, 1.94 mmol) in THF (5 mL) was stirred at ambient temperature for 24 h. The excess of solvent was removed in vacuo, and the crude product thus obtained was purified using column chromatography. 1-(3 -Deoxy-2 -O,4 -C-methylenethymidin-3 -yl)-4-(1-(5-deoxy-2-O,4C-methylene-β-D-xylofuranosyl)thymin-5-yl)-1,2,3-triazole (2). It was obtained as light yellow solid (0.77 g, 92%, using method A), (0.63 g, 75%, using method B). Rf = 0.3 (10% methanol in chloroform). M. Pt.: decomposes at 202–204◦ C. [α]D 35 = + 28.9 (c 0.1, MeOH); IR (KBr)νmax: 3405, 2930, 2816, 1700, 1471, 1275, 1110, 1060, 1016, 992, 895, and 805 cm−1; 1H NMR (DMSO-d 6 , 300 MHz): δ 11.03 (2H, brs), 8.11 (1H, s), 7.70 (1H, s), 7.59 (1H, s), 5.83 (1H, brs), 5.64 (1H, s), 5.44 (2H, brs), 5.08 (1H, s), 4.80 (1H, s), 4.19–3.83 (6H, m,), 3.61 (1H, d, J = 7.5 Hz), 3.61 (1H, d, J = 7.5 Hz), 3.25 (2H, q, J = 7.2 Hz), 1.79 (3H, s), 1.77 (3H, s); 13 C NMR (DMSO-d 6 , 75.5 MHz): δ 164.0, 163.9, 150.0, 150.0, 141.1, 137.1, 134.5, 123.9, 108.5, 105.9, 90.2, 88.1, 86.1, 79.1, 77.3, 73.3, 72.6, 70.9, 59.3, 56.6, 22.8, 12.4. HRMS: m/z calculated for [C24 H27 N7 O10 +H+] 574.1892, observed 574.1885. 1-(3 -Deoxy-2 -O,4 -C-methylenethymidin-3 -yl)-4-(9-(5-deoxy-2-O,4C-methylene-β-D-xylofuranosyl)-N 6-benzoyladenin-5-yl)-1,2,3-triazole (3). It was obtained as white solid (0.90 g, 90%, using method A), (0.65 g, 65%, using method B). M. Pt.: 199–201◦ C; [α]D 30 = −1.85 (c 0.1, MeOH); IR (KBr) νmax: 3400, 2924, 2853, 1701,1458, 1251, 1212, 1111, 1055, 988, 895, 847, 798, 640, and 581 cm−1; 1H NMR (DMSO-d6 , 400 MHz): δ 11.38 (1H, brs), 11.13 (1H, brs), 8.68 (1H, s), 8.46(1H, s), 8.10 (1H, s), 7.99 (2H, d, J = 7.2 Hz), 7.67 (1H, s), 7.60 (1H, t, J = 8.0 Hz), 7.50 (2H, t, J = 8.0 Hz), 6.07 (1H, s), 6.07 (1H, brs), 5.62 (1H, s), 5.43 (1H, t, J = 5.6 Hz), 5.06 (1H, s), 4.78 (1H, s), 4.55 (1H, d, J = 2.4 Hz), 4.13–4.12 (1H, m), 4.02–3.76 (5H, m), 3.55 (1H, d, J = 8.8 Hz), 3.25 (2H, s), 1.76 (3H, s); 13C NMR (CDCl3 , 100.6 MHz): δ 165.6, 164.0, 151.6, 150.1, 149.9, 143.5, 141.3, 134.7, 133.3, 132.5, 128.5, 125.3, 124.0, 108.7, 90.3, 87.7, 86.5, 86.2, 79.2, 77.9, 73.1, 71.0, 59.4, 56.7, 23.0, 12.4; HRMS: m/z calculated for [C31 H30 N10 O9 +H]+ 687.2270, observed 687.2243. 1-(3 -Deoxy-2 -O,4 -C-methylenethymidin-3 -yl)-4-(1-(5-deoxy-2-O,4C-methylene-β-D-xylofuranosyl)-N 4-benzoylcytosin-5-yl)-1,2,3-triazole (4). It was obtained as white solid (0.89 g, 92%, using method A), (0.67 g, 70%, using method B). M. Pt.: 220–222◦ C; [α]D 30 = +88.50 (c 0.1, MeOH); IR (KBr) νmax: 3422, 2928, 1685, 1481, 1313, 1264, 1109, 1051, 896, 790, 707 and 582 cm−1; 1H NMR (DMSO-d6 , 400 MHz): δ 11.4 (1H, s), 11.20 (1H,

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brs), 8.24 (2H, d, J = 7.2 Hz), 8.14 (1H, s), 8.00 (2H, d, J = 7.6 Hz), 7.73 (1H, d, J = 1.2 Hz), 7.63 (1H, t, J = 7.2 Hz), 7.51 (1H, t, J = 7.2 Hz), 7.32 (1H, d, J = 7.2 Hz), 5.87 (1H, brs), 5.67 (1H, s), 5.55 (1H, s), 5.49 (1H, brs), 5.11 (1H, s), 4.83 (1H, s), 4.34 (1H, d, J = 2.0 Hz), 4.10–3.86 (5H, m), 3.74 (1H, d, J = 8.0 Hz), 3.62 (1H, d, J = 8.8 Hz), 3.33–3.27 (2H, m), 1.81 (3H, s); 13C NMR (CDCl3 , 100.6 MHz): δ 168.1, 165.1, 163.2, 155.5, 150.5, 147.2, 141.8, 135.4, 133.6, 133.1, 129.2, 128.4, 124.6, 109.6, 95.5, 90.5, 89.7, 88.9, 86.6, 79.3, 77.9, 74.0, 73.6, 71.2, 66.6, 59.8, 56.6, 23.4, 22.9, 12.6; HRMS m/z calculated for [C30 H30 N8 O10 +H]+ 663.2158, observed 663.2142.

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FUNDING We are grateful to the University of Delhi for providing financial support under DU-DST Purse Grant and R & D Scheme. SS, SKS, and VKS thank CSIR and PM thanks UGC, New Delhi for the awards of Junior/Senior Research Fellowships. REFERENCES 1. Stephenson, M.L.; Zamecnik, P.C. Inhibition of rous sarcoma viral RNA translation by specific oligodeoxyribonucleotide. Proc. Natl. Acad. Sci. USA. 1978, 75, 285–288. 2. Campbell, M.A.; Wengel, J. Locked vs. unlocked nucleic acids (LNA vs. UNA): contrasting structures work towards common therapeutic goals. Chem. Soc. Rev. 2011, 40, 5680–5689. 3. Snead, N.M.; Rossi, J.J. RNA interference trigger variants: getting the most out of RNA for RNA interference-based therapeutics. Nucleic Acid Ther. 2012, 22, 139–146. 4. Sharma, V.K.; Rungta, P.; Prasad, A.K. Nucleic acid therapeutics: basic concepts and recent developments. RSC Adv. 2014, 4, 16618–16631. 5. (a) Braasch, D.A.; Corey, D.R. Locked nucleic acid (LNA): fine tuning the recognition of DNA and RNA. Chem. Biol. 2001, 8, 1–7. (b) Rajwanshi, V.K.; H˚akansson, A.E.; Kumar, R.; Wengel, J. Highaffinity nucleic acid recognition using ‘LNA’ (locked nucleic acid, β-D-ribo configured LNA), ‘xyloLNA’ (β-D-xylo configured LNA) or ‘α-L-LNA’ (α-L-ribo configured LNA). Chem. Commun. 1999, 2073–2074. (c) Rajwanshi, V.K.; H˚akansson, A.E.; Sørensen, M.D.; Pitsch, S.; Singh, S.K.; Kumar, R.; Neilsen, P.; Wengel, J. The eight stereoisomers of LNA (Locked Nucleic Acid): a remarkable family of strong RNA binding molecules. Angew.Chem., Int. Ed. 2000, 39, 1656–1659. (d) Lebreton, J.; Escudier, J.; Arzel, L.; Len, C. Synthesis of bicyclonucleosides having a C-C bridge. Chem. Rev. 2010, 110, 3371–3418. 6. (a) Mickelfield, J. Backbone modification of nucleic acids: synthesis, structure and therapeutic applications. Curr. Med. Chem. 2001, 8, 1157–1161. (b) Isobe, H.; Fujino, T. Triazole-linked analogues of DNA and RNA (TLDNA and TLRNA): synthesis and functions. Chem. Rec. 2014, 14, 41–51. (c) Kumar, P.; Hornum, M.; Nielsen, L.J.; Enderlin, G.; Andersen, N.K.; Len, C.; Herv´e,G.; Sartori, G.; Nielsen, P. High-affinity RNA targeting by oligonucleotides displaying aromatic stacking and amino groups in the major groove. comparison of triazoles and phenyl substituents. J. Org. Chem. 2014, 79, 2854–2863. (d) Chandrasekhar, S.; Srihari, P.; Nagesh, C.; Kiranmai, N.; Nagesh, N.; Idris, M.M. Synthesis of readily accessible triazole-linked dimer deoxynucleoside phosphoramidite for solid-phase oligonucleotide synthesis. Synthesis 2010, 21, 3710–3714. 7. (a) Robins, M.J.; Doboszewski, B.; Timoshchuk, V.A.; Peterson, M.A. Glucose-derived 3 (carboxymethyl)-3 -deoxyribonucleosides and 2 ,3 -lactones as synthetic precursors for amide-linked oligonucleotide analogues. J. Org. Chem. 2000, 65, 2939–2945. (b) Lauristen, A.; Wengel, J. Oligodeoxynucleotides containing amide-linked LNA-type dinucleotides: synthesis and high-affinity nucleic acid hybridization. Chem. Commun. 2002, 530–531.

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8. Pallan, P.S.; Matt, P.; Wilds, C.J.; Altmann, K.H.; Egli, M. RNA-binding affinities and crystal structure of oligonucleotides containing five-atom amide-based backbone structures. Biochemistry 2006, 45, 8048–8057. 9. (a) Gogoi, K.; Gunjal, A.D.; Phalgune, U.D.; Kumar, V.A. Synthesis and RNA binding selectivity of oligonucleotides modified with five atom thioacetamido nucleic acid backbone structures. Org. Lett. ` I.E.; Bruice, T.C. Incorporation of positively 2007, 9, 2697–2700. (b) Jain, M.L.; Bruice, P.Y.; Szabo, charged linkages into DNA and RNA backbones: a novel strategy for antigene and antisense agents. Chem. Rev. 2012, 112, 1284–1309. 10. (a) Amblard, F.; Cho, J.H.; Schinazi, R.T. Cu(I)-catalyzed huisgen azide-alkyne 1,3-dipolar cycloaddition reaction in nucleoside, nucleotide and oligonucleotide chemistry. Chem Rev. 2009, 109, 4207–4220. (b) Varizhuk, A.; Chizhov, A.; Florentiev, V. Synthesis and hybridization data of oligonucleotide analogs with triazole internucleotide linkages, potential antiviral and antitumor agents. Bioorganic Chem. 2011, 39, 127–131. (c) Varizhuk, A.M.; Kaluzhny, D.N.; Novikov, R.A.; Chizhov, A.O.; Smirnov, I.P.; Chuvilin, A.N.; Tatarinova, O.N.; Fisunov, G.Y.; Pozmogova, G.E.; Florentiev, V.L. Synthesis of triazole-linked oligonucleotides with high affinity to DNA complements and an analysis of their compatibility with biosystems. J. Org. Chem. 2013, 78, 5964–5969. 11. Lucas, R.; Neto, V.; Bovazza, A.H.; Zerrouki, R.; Granet, R.; kravsz, P.; Champavier, Y. Microwaveassisted synthesis of a triazole-linked 3 –5 dithymidine using click chemistry. Tetrahedron Lett. 2008, 49, 1004–1007. 12. Isobe, H.; Fujino, T.; Yamazaki, N.; Niekowski, M.G.; Nakamura, E. Triazole-linked analogue of deoxyribonucleic acid (TLDNA): design, synthesis, and double-strand formation with natural DNA. Org. Lett. 2008, 10, 3729–3732. 13. Fujino, T.; Tsunaka, N.; Nieckowski, M.G.; Nakanishi, W.; Iwamoto, T.; Nakamura, E.; Isobe, H. Synthesis and structures of deoxyribonucleoside analogues for triazole-linked DNA (TLDNA). Tetrahedron Lett. 2010, 51, 2036–2038. 14. (a) Singh, S.K.; Sharma, V.K.; Bohra, K.; Olsen, C.E.; Prasad, A.K. Synthesis of triazole-linked LNAbased nonionic nucleoside dimers using Cu(I) catalysed ‘click’ reaction. Curr. Org. Synth. 2014, 11(5), 757–766. (b) Bohra, K.; Srivastava, S.; Olsen, C.E.; Prasad, A.K. Synthesis of sugar modified triazole-linked nucleoside dimers. Trends Carbohyd. Res. 2014, 6, 27–32. 15. Kolb, H.C.; Sharpless, K.B. The growing impact of click chemistry on drug discovery. Drug Discov. Today 2003, 8, 1128–37. (b) Meldal, M.; Tornøe, C.W. Cu-catalyzed azide- alkyne cycloaddition. Chem. Rev. 2008, 108, 2952–3015. 16. Sharma, V.K.; Singh, S.K.; Bohra, K.; Reddy, C.S.L.; Khatri, V.; Olsen, C.E.; Prasad, A.K. Design and synthesis of LNA-based mercaptoacetamido-linked nucleoside dimers. Nucleosides Nucleotides Nucleic Acids. 2013, 32, 256–272. 17. Youssefyeh, R.D.; Verheyden, J.P.H.; Moffatt, J.G. 4 -Substituted nucleosides. 4. synthesis of some 4 -hydroxymethyl nucleosides. J. Org. Chem. 1979, 44, 1301–1309. ¨ 18. Vorbruggen, H.; Lagoja, I.M.; Herdewijn, P. Synthesis of ribonucleosides by condensation using trimethylsilyl triflate. Curr. Protocols Nucl. Acid Chem. 2007, 1.13. 19. (a) Lucas, R.; Neto, V.; Bouazza, A.H.; Zerrouki, R.; Granet, R.; Krausz, P.; Champavier, Y. Microwaveassisted synthesis of a triazole-linked 3 –5 dithymidine using click chemistry. Tetrahedron Lett. 2008, 49, 1004–1007. (b) Bock, V.D.; Hiemstra, H.; Maarseveen, J.H. CuI-catalyzed alkyne–azide “click” cycloadditions from a mechanistic and synthetic perspective. Eur. J. Org. Chem. 2006, 51–68. (c) Himo, F.; Lovell, T.; Hilgraf, R.; Rostovtsev, V.V.; Noodleman, L.; Sharpless, K.B.; Fokin, V.V. Copper(I)catalyzed synthesis of azoles. DFT study predicts unprecedented reactivity and intermediates. J. Am. Chem. Soc. 2005, 127, 210–216.

Design and Synthesis of Triazole-Linked xylo-Nucleoside Dimers.

Three triazole-linked nonionic xylo-nucleoside dimers T(L)-t-T(xL), T(L)-t-A(BzxL) and T(L)-t-C(BzxL) have been synthesized for the first time by Cu(I...
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