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Synthesis 2010(21): 3710-3714  
DOI: 10.1055/s-0030-1258243
PAPER
© Georg Thieme Verlag Stuttgart ˙ New York

Synthesis of Readily Accessible Triazole-Linked Dimer Deoxynucleoside Phosphoramidite for Solid-Phase Oligonucleotide Synthesis

Srivari Chandrasekhar*a, Pabbaraja Sriharia, Chidalla Nagesha, Nayani Kiranmaia, Narayana Nageshb, M. Mohammed Idris*b
a Organic Chemistry Division - I, Indian Institute of Chemical Technology, Hyderabad 500 007, India
Fax: +91(40)27160512; e-Mail: srivaric@iict.res.in;
b Centre for Cellular and Molecular Biology, Hyderabad 500 007, India
e-Mail: idris@ccmb.res.in.;

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Publication History

Received 25 June 2010
Publication Date:
07 September 2010 (online)

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Abstract

A concise preparation of triazole-linked deoxynucleoside phosphoramidite for its direct use in solid-phase oligonucleo­tide synthesis is reported. This dimer has successfully been utilized in solid-phase synthesis to make the 10- and 12-mer oligomers.

Key words

nucleotides - triazole - click - oligomer - solid-phase synthesis

Modified nucleosides/nucleotides continue to attract significant focus in medical applications for gene silencing such as, antisense, antigene, and RNA interference. [¹] As naturally occurring oligonucleotides suffer from degradation due to endo- and exonucleases, modifications have become necessary to enhance the stability and also improve pharmacokinetic properties from a therapeutic point of view. Since the introduction of antisense agents, diverse analogues have been developed that include, modifications­ in the phosphate backbone (see Figure  [¹] ); phosphate analogues such as phosphorothioate A, phosphoramidate­, methyl phosphonate, [²] and nucleotide borano phosphates B; [³] complete replacement of the phosphodiester linkage by more stable functionalities like amide C, [4] amine [5] and heterocycles D, E, [6] F; [7] modifications in nucleoside bases; [8] derivatization/substitution of the sugar moiety [9] and complete replacement of furano phosphate moiety to result in PNA; [¹0] and replacement of the sugar moiety with morpholine etc. [¹¹] In parallel, the latest, innovative reactions such as the click reaction, have been well utilized to derive modified nucleosides. [¹²] Our own interest in the click reaction has encouraged us to utilize it for the synthesis of new scaffolds from Baylis-Hillman­ adducts to result in triazoles [¹³] and tetrazoles [¹4] that displayed potency when screened against TNF-α inhibition studies towards anti-arthritis.

Recently, Isobe et al. have accomplished the synthesis of a 10-mer TLDNA oligomer chain with click chemistry wherein the phosphate group has been completely replaced with by a triazole. [7] This new analogue was found to form a stable double strand with the complementary strand of natural DNA. The linkage of sugars through the triazole moieties throughout the chain makes it a limiting factor for further exploration. To overcome this, we initiated synthesis of a dimer nucleoside phosphoramidite with a triazole linkage that can be utilized directly for oligonucleotide synthesis on a solid support and substituted/linked up at the requisite site(s) in the chain. In continuation to our research focused on click chemistry, [¹5] we herein wish to report the synthesis of a dimer nucleoside phosphoramidite 1, wherein the two monomer nucleoside units are interlinked through a triazole moiety rather than the regular phosphate ester linkage 1′ (Figure  [²] ).

Figure 1 Some modified thymidine dinucleotides

Figure 2 Thymidine dimers

Even though the replacement of the phosphodiester moiety with heterocycles, such as imidazole and tetrazoles, is known, [6] replacement with the triazole moiety has not been well explored. [¹²] [¹6] We started to design a new dinucleoside phosphoramidite 1 with the sugar part of the nucleotide unaltered. Retrosynthetic analysis for 1 revealed two key fragments, azide 2 (AZT) and acetylene 3. Compound 3 was easily accessible from commercially available thymidine 4 in five steps (Scheme  [¹] ).

Scheme 1 Retrosynthesis for modified dimer nucleoside phosphoramidite 1

For the synthesis, we started with thymidine 4 which was protected as its bis(silyl ether) 5 at the 3′- and 5′-positions. Selective primary silyl deprotection was achieved with 10-camphorsulfonic acid [¹7] to give the free primary alcohol 6. The alcohol 6 was oxidized with Dess-Martin periodinane and was subjected to Corey-Fuchs protocol [¹8] to give dibromomethylene 7. Compound 7 was treated with freshly generated ethylmagnesium bromide to give the monomer 3 with a terminal acetylene moiety. [¹9] Compound 3 was subjected to cycloaddition with commercially available AZT 2 in the presence of copper metal in ethanol at reflux temperature to give the nucleoside dimer 8 in 75% yield. [¹5a] Our next goal was to protect the free primary alcohol (5′-OH) with 4,4′-dimethoxytrityl chloride and convert the protected hydroxy group at the 3′-position to a phosphoramidite to obtain the readily accessible dimer nucleoside phosphoramidite. Thus, we proceeded with the protection of free 5′-OH group in 8 as the corresponding dimethoxytrityl ether 9 with 4,4′-dimethoxytrityl chloride in the presence of pyridine and 4-(dimethylamino)pyridine. Tetrabutylammonium fluoride mediated deprotection of silyl ether 9 afforded 10 which was converted into the target phosphoramidite 1 using 2-cyanoethyl diisopropylchlorophosphoramidite in the presence of N,N-diisopropylethylamine in anhydrous dichloromethane at room temperature. [¹6f] Thus, we have accomplished the synthesis of the target dimer deoxynucleoside phosphoramidite 1 (Scheme  [²] ).

Scheme 2 Synthesis of modified dinucleotide 1 (triazole-linked dimer)

The synthesized dinucleoside 1 was a suitably protected dimer that can be utilized directly for solid-phase oligonucleotide synthesis. We have successfully demonstrated the utilization of compound 1 for the synthesis of two oligomers of length 10-mer poly mix GC(TT*)AG(TT*)TA and 12-mer (poly T, TT(TT*)TT(TT*)TTTT which were synthesized in the oligo synthesizer (Scheme  [³] ). These oligos were characterized by MALDI analysis with m/z 2975.697 for 10-mer poly mix, GC(TT*)AG(TT*)TA and m/z 3530.752 for 12-mer poly T, TT(TT*)TT(TT*)TTTT.

Scheme 3 Application of modified dimer in oligomer synthesis

In conclusion, a triazole-linked modified dinucleoside phosphoramidite synthesis and its incorporation into long-chain oligonucleotides on solid phase were demonstrated. Further investigations on the properties of this dimer nucleoside phosphoramidite and its application in long-chain oligomer synthesis and also the synthesis of other dinucleotide combinations are in progress.

All the reagents employed were obtained commercially from M/s. Aldrich and used without further purifications unless otherwise stated. For anhydrous reactions, solvents were dried by literature procedures; removal of solvent was performed under reduced pressure using a rotary evaporator. All reactions requiring anhydrous conditions were carried out in oven-dried glassware under N2. All reactions and fractions from column chromatography were monitored by TLC using plates with a UV fluorescent indicator (normal silica gel, Merck 60 F254). ¹H NMR spectra were recorded at 300 or 400 MHz [relative to the solvent residual signal in CDCl3 (δ = 7.26) or to TMS], and ¹³C NMR spectra were recorded at 75 MHz/100 MHz at r.t. [relative to the solvent signal CDCl3 (δ = 77.00) unless otherwise noted]. One or more of the following methods were used for visualization: UV absorption by fluorescence quenching; I2 staining; phosphomolybdic acid/Ce(SO4)2/H2SO4/H2O (10 g:1.25 g:12 mL:238 mL) spray; anisaldehyde spray (EtOH-AcOH-H2SO4-anisaldehyde; 200 mL:2 mL:6.35 mL:3 mL). Column chromatography was performed using 60-120 mesh silica gel.

Oligonucleotide Synthesis

The solid phase oligonucleotide synthesis was performed on an ABI-394 DNA synthesizer (USA) following standard procedures. Detritylation was achieved using 2-3% trichloroacetic acid in CH2Cl2. The incoming base was first allowed to react with weak acid tetrazole to form an active tetrazolyl phosphoramidite intermediate which reacts with the 5′-hydroxy group of the recipient. Unreacted free hydroxy groups were capped by Ac2O and N-methylimidazole in MeCN. Stabilization of the phosphate linkage between two bases was achieved by treating the resin-supported oligomer with I2 in THF-H2O. After the completion of the chain synthesis, the mixture was incubated at 55 ˚C for 16 h to cleave the resin as well as remove the protection groups. The modified base phosphoramidite as well as DMTr+ on oligonucleotides were purified by passing through the DMTr+ Glen-Pak Cartridges (Glen Research Inc, USA) and they were characterized by mass spectroscopy and gel electrophoresis.

1-{(2 R ,4 S ,5 R )-4-( tert -Butyldimethylsilyloxy)-5-[( tert -butyldi­methylsilyloxy)methyl]tetrahydrofuran-2-yl}-5-methylpyrimidine-2,4(1 H ,3 H )-dione (5)

To the soln of thymidine 4 (3.0 g, 12.38 mmol) in CH2Cl2 (20 mL) at 0 ˚C under N2 was added imidazole (2.52 g, 37.15 mmol) and the mixture was stirred for 20 min. TBSCl (4.65 g, 30.96 mmol) in CH2Cl2 (10 mL) was added dropwise and the mixture was stirred at r.t. for 1 h. After completion of the reaction, the mixture was quenched with H2O (20 mL) and extracted with CH2Cl2 (3 × 20 mL). The combined organic layers were washed with brine (30 mL), dried (anhyd Na2SO4), and concentrated in vacuo. The residue was subjected to column chromatography (silica gel, EtOAc-hexane, 1:5) to yield 5 (5.8 g, 99%) as a white solid; mp 141-142 ˚C; R f = 0.80 (EtOAc-hexane, 1:1).

¹H NMR (300 MHz, CDCl3): δ = 8.66 (s, 1 H), 7.4 (d, J = 0.9 Hz, 1 H), 6.25 (dd, J = 7.3, 6.0 Hz, 1 H), 4.43-4.50 (m, 1 H), 3.72-3.98 (m, 3 H), 2.24 (ddd, J = 13.1, 5.89, 3.03 Hz, 1 H), 1.98 (ddd, J = 13.1, 7.5, 6.5 Hz, 1 H), 1.91 (d, J = 0.9 Hz, 3 H), 0.94 (s, 9 H), 0.91 (s, 9 H), 0.11 (d, J = 0.9 Hz, 6 H), 0.08 (d, J = 3.4 Hz, 6 H).

¹³C NMR (75 MHz, CDCl3): δ = 163.5, 150.1, 135.4, 110.7, 87.8, 84.8, 72.2, 62.9, 41.3, 25.9, 25.7, 18.3, 17.9, 12.5, -4.6, -4.8, -5.3.

HRMS: m/z calcd [M + Na]+ for C22H42N2O5NaSi2: 493.2516; found: 493.2529.

1-[(2 R ,4 S ,5 R )-4-( tert -Butyldimethylsilyloxy)-5-(hydroxymethyl)tetrahydrofuran-2-yl]-5-methylpyrimidine-2,4(1 H ,3 H )-dione (6)

CSA (0.812 g, 3.49 mmol) was added portionwise to a stirred soln of 5 (2.78 g, 5.91 mmol) in CH2Cl2-MeOH (1:1, 20 mL) at 0 ˚C under N2 and the mixture was stirred at 0 ˚C for 16 h. The mixture was quenched with aq NaHCO3 (20 mL) at 0 ˚C and stirred for 30 min. The solvents were evaporated in vacuo and the aqueous layer was extracted with CH2Cl2 (3 × 20 mL). The combined organic layers were washed with brine (20 mL), dried (anhyd Na2SO4), and concentrated in vacuo. The residue was subjected to column chromatography (silica gel, EtOAc-hexane, 2:3) to yield 6 (1.45 g, 69%) as a white solid; mp 89-91 ˚C; R f = 0.50 (EtOAc).

¹H NMR (300 MHz, CDCl3): δ = 9.80 (br s, 1 H), 7.36 (s, 1 H), 6.07 (t, J = 6.6 Hz, 1 H), 4.45-4.54 (m, 1 H), 3.82-3.94 (m, 2 H), 3.64-3.76 (m, 1 H), 3.10 (br s, 1 H), 2.27-2.42 (m, 1 H), 2.21-2.25 (m, 1 H), 1.88 (s, 3 H), 0.90 (s, 9 H), 0.09 (s, 6 H).

¹³C NMR (75 MHz, CDCl3): δ = 164.2, 150.6, 137.2, 111.1, 87.7, 86.9, 71.7, 62.1, 40.7, 25.9, 18.1, 12.6, -4.5, -4.6.

HRMS: m/z calcd [M]+ for C16H28N2O5Si: 356.1776; found: 356.1767.

1-[(2 R ,4 S ,5 R )-4-( tert -Butyldimethylsilyloxy)-5-(2,2-dibromovinyl)tetrahydrofuran-2-yl]-5-methylpyrimidine-2,4(1 H ,3 H )-dione (7)

To a stirred soln of 6 (1.38 g, 3.87 mmol) in CH2Cl2 (30 mL) was added Dess-Martin periodinane (1.78 g, 4.21 mmol) and the mixture was stirred at r.t. for 30 min. The reaction was quenched with sat. aq Na2S2O3 (10 mL) at 0 ˚C. The mixture was diluted with CH2Cl2 (10 mL) and the aqueous layer was extracted with CH2Cl2 (2 × 10 mL). The combined organic layers were washed with sat. aq NaHCO3 (10 mL) and brine (10 mL), dried (anhyd Na2SO4), and concentrated in vacuo to give crude aldehyde (1.30 g) that was utilized directly in the next reaction without further purification. To the stirred soln of Ph3P (3.7 g, 14.12 mmol) in CH2Cl2 (10 mL) at 0 ˚C under N2 was added CBr4 (2.34 g, 7.06 mmol) dissolved in CH2Cl2 (10 mL) slowly and the reaction was stirred at this temperature for 30 min and then warmed to r.t. The mixture was cooled to 0 ˚C and Et3N (1.07 g, 10.59 mmol) was added and the mixture was allowed to warm to r.t. for 15 min. The mixture was recooled to 0 ˚C and to this was added the above aldehyde (1.38 g, 3.53 mmol) dissolved in CH2Cl2 (10 mL). When the reaction was complete, the mixture was quenched with H2O (30 mL) and stirred for 30 min, the organic layer was separated and the aqueous layer was extracted with CH2Cl2 (3 × 20 mL). The combined organic layers were washed with brine (20 mL), dried (anhyd Na2SO4), and concentrated in vacuo. The residue was subjected to column chromatography (silica gel, EtOAc-hexane, 1:4) to yield 7 (1.62 g, 82%) as a light brown solid; mp 149-151 ˚C; R f  = 0.50 (EtOAc-hexane, 4:6).

¹H NMR (300 MHz, CDCl3): δ = 8.44 (br s, 1 H), 7.06 (d, J = 1.1 Hz, 1 H), 6.50 (d, J = 8.5 Hz, 1 H), 6.01 (t, J = 6.6 Hz, 1 H), 4.52 (dd, J = 8.5, 3.9 Hz, 1 H), 4.29-4.36 (m, 1 H), 2.19-2.40 (m, 2 H), 1.95 (d, J = 1.1 Hz, 3 H), 0.91 (s, 9 H), 0.12 (s, 3 H), 0.10 (s, 3 H).

¹³C NMR (75 MHz, CDCl3): δ = 163.9, 150.4, 136.1, 135.5, 111.4, 87.1, 86.7, 75.5, 73.4, 40.4, 25.8, 18.1, 12.8, -4.4, -4.6.

HRMS: m/z calcd [M + Na]+ for C17H26Br2N2O4NaSi: 530.9897; found: 530.9926.

1-[(2 R ,4 S ,5 R )-4-( tert -Butyldimethylsilyloxy)-5-ethynyltetrahydrofuran-2-yl]-5-methylpyrimidine-2,4(1 H ,3 H )-dione (3)

EtBr (3.04 g, 27.93 mmol) was added to a suspension of Mg (0.50 g, 18.62 mmol) in anhyd THF (20 mL) dropwise. After generation of EtMgBr, the mixture was cooled to -20 ˚C and to this was added compound 7 (0.95 g, 1.86 mmol) in THF (5 mL). After consumption of the starting material, the mixture was quenched with sat. aq NH4Cl (10 mL) at 0 ˚C, the mixture was diluted with H2O (10 mL), and the aqueous layer was extracted with EtOAc (2 × 20 mL). The combined organic layers were washed with brine (10 mL), dried (anhyd Na2SO4), and concentrated in vacuo. The residue was subjected to column chromatography (silica gel, EtOAc-hexane, 1:4) to yield 3 (0.45 g, 70%) as a white solid; mp 158-160 ˚C; R f = 0.50 (EtOAc-hexane, 1:1).

¹H NMR (400 MHz, CDCl3): δ = 8.98 (br s, 1 H), 7.52 (s, 1 H), 6.42 (dd, J = 5.8, 8.1 Hz, 1 H), 4.58 (s, 1 H), 4.51 (d, J = 3.66, 4.3 Hz, 1 H), 2.75 (d, J = 1.5 Hz, 1 H), 2.43 (ddd, J = 4.4, 5.8, 13.1 Hz, 1 H), 2.16 (ddd, J = 4.4, 8.1, 13.1 Hz, 1 H), 1.94 (s, 3 H), 1.91 (s, 9 H), 0.12 (s, 6 H).

¹³C NMR (100 MHz, CDCl3): δ = 163.7, 150.3, 135.5, 111.1, 86.8, 80.5, 77.6, 76.9, 40.5, 25.6, 17.9, 12.7, -4.8, -4.9.

HRMS: m/z calcd [M + Na]+ for C17H26N2O4NaSi: 373.1556; found: 373.1559.

1-[(2 R ,4 S ,5 R )-4-( tert -Butyldimethylsilyloxy)-5-(1-{(2 S ,3 S ,5 R )-2-(hydroxymethyl)-5-[5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2 H )-yl]tetrahydrofuran-3-yl}-1 H -1,2,3-triazol-4-yl)tetrahydrofuran-2-yl]-5-methylpyrimidine-2,4(1 H ,3 H )-dione (8)

Cu(0) (20 mg) was added to a stirred soln of AZT 2 (0.431 g, 1.61 mmol) and 3 (0.565 g, 1.61 mmol) in EtOH (20 mL). Sat. aq CuSO4 (0.5 mL) was added to the mixture and it was refluxed for 12 h. After the completion of the reaction, the mixture was filtered through Celite and washed with CHCl3, and the filtrate was concentrated in vacuo. The residue was subjected to column chromatography (silica gel, MeOH-CHCl3, 2:98) to yield 8 (0.74 g, 75%) as a white solid; mp 139-141 ˚C; R f  = 0.30 (EtOAc).

¹H NMR (300 MHz, CDCl3): δ = 9.25 (s, 1 H), 9.21 (s, 1 H), 7.89 (s, 1 H), 7.59 (d, J = 1.1 Hz, 1 H), 7.49 (d, J = 0.95 Hz, 1 H), 6.28 (t, J = 6.8 Hz, 1 H), 6.22 (t, J = 6.2 Hz, 1 H), 5.42-5.51 (m, 1 H), 4.98 (d, J = 3.2 Hz, 1 H), 4.70-4.77 (m, 1 H), 4.42-4.48 (m, 1 H), 3.99-4.08 (m, 1 H), 3.74-3.85 (m, 1 H), 3.41-3.50 (m, 1 H), 2.91-3.08 (m, 2 H), 2.64-2.72 (m, 1 H), 2.36 (ddd, J = 3.7, 6.2, 13.4 Hz, 1 H), 1.94 (d, J = 0.75 Hz, 3 H), 1.91 (d, J = 0.75 Hz, 3 H), 0.86 (s, 9 H), 0.03 (s, 3 H), 0.01 (s, 3 H).

¹³C NMR (75 MHz, CDCl3): δ = 164.0, 150.8, 150.5, 146.1, 137.7, 137.6, 123.2, 111.1, 88.3, 87.5, 85.3, 81.0, 76.1, 61.1, 58.9, 39.8, 37.6, 29.6, 25.6, 17.9, 12.5, 12.4, -4.8.

HRMS: m/z calcd [M]+ for C27H39N7O8Si: 617.2629; found: 617.2638.

1-[(2 R ,4 S ,5 S )-5-{[Bis(4-methoxyphenyl)(phenyl)meth­oxy]methyl}-4-(4-{(2 R ,3 S ,5 R )-3-( tert -butyldimethylsilyloxy)-5-[5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2 H )-yl]tetrahydrofuran-2-yl}-1 H -1,2,3-triazol-1-yl)tetrahydrofuran-2-yl]-5-methylpyrimidine-2,4(1 H ,3 H )-dione (9)

Nucleoside 8 (0.500 g, 0.809 mmol) was co-evaporated with dry pyridine (2 × 10 mL) and dried under vacuum for 30 min. To a soln of dried 8 (0.500 g, 0.809 mmol) in dry pyridine (40 mL) was added DMAP (0.217 g, 1.78 mmol) and 4,4′-dimethoxytrityl chloride (0.329 g, 0.971 mmol) at r.t. under argon. The mixture was stirred at r.t. for 20 h, at which time TLC indicated that the reaction was not complete (only 20% conversion). To the mixture was added additional DMAP (0.217 g, 1.78 mmol) and 4,4′-dimethoxytrityl chloride (0.329 g, 0.971 mmol). The mixture was stirred at r.t. until the reaction was complete (˜6 h). The solvent was removed, and the residue was purified by chromatography (silica gel, MeOH-CHCl3, 2:98) to give 9 (0.591g, 80%) as a light yellow foam; mp 162-163 ˚C; R f  = 0.45 (MeOH-CHCl3, 1:9).

¹H NMR (300 MHz, CDCl3): δ = 8.94 (br s, 1 H), 8.75 (br s, 1 H), 7.69 (d, J = 1.1Hz, 1 H), 7.66 (d, J = 0.7 Hz, 1 H), 7.45 (s, 1 H,), 7.37-7.43 (m, 2 H), 7.24-7.35 (m, 7 H), 6.84 (dd, J = 8.8, 9.0 Hz, 4 H), 6.42-6.53 (m, 2 H), 5.32-5.41 (m, 1 H), 4.88 (d, J = 2.8 Hz, 1 H), 4.63-4.69 (m, 1 H), 4.39-4.46 (m, 1 H), 3.79 (s, 6 H), 3.70 (dd, J = 11.4, 10.9 Hz, 1 H), 3.35 (dd, J = 10.9, 10.7 Hz, 1 H), 3.03-3.13 (m, 1 H), 2.58-2.82 (m, 2 H), 2.31-2.41 (m, 1 H), 1.87 (s, 3 H), 1.60 (s, 3 H), 0.87 (s, 9 H), 0.07 (s, 6 H).

¹³C NMR (75 MHz, CDCl3): δ = 163.3, 158.8, 150.4, 150.1, 146.2, 143.9, 136.6, 135.3, 134.9, 134.8, 130.0, 128.1, 127.9, 127.3, 122.2, 113.3, 111.6, 111.2, 87.1, 86.2, 85.2, 83.6, 80.6, 77.1, 76.1, 62.0, 59.7, 55.2, 40.0, 38.3, 25.6, 17.9, 12.5, 12.0, 0.9. -4.8, -4.8.

HRMS: m/z calcd [M + H]+ for C48H58N7O10Si: 920.4009; found: 920.4003.

1-[(2 R ,4 S ,5 S )-5-{[Bis(4-methoxyphenyl)(phenyl)meth­oxy]methyl}-4-(4-{(2 R ,3 S ,5 R )-3-hydroxy-5-[5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2 H )-yl]tetrahydrofuran-2-yl}-1 H -1,2,3-triazol-1-yl)tetrahydrofuran-2-yl]-5-methylpyrimidine-2,4(1 H ,3 H )-dione (10)

A 1 M soln of TBAF (0.11 g, 0.43 mmol) was added to a stirred soln of 9 (0.40 g, 0.43 mmol) in THF (5 mL) at 0 ˚C under N2 and the mixture was stirred for 1 h. The reaction was quenched with sat. NaHCO3 and stirred for 10 min. The aqueous layer was extracted with EtOAc (3 × 5 mL), the combined organic layers were washed with brine (5 mL), dried (anhyd Na2SO4), and concentrated in vacuo. The residue was subjected to column chromatography (silica gel, MeOH-CHCl3, 5:95) to yield 7 (0.35 g, 99%) as a light foamy white solid; mp 123-125 ˚C; R f  = 0.40 (MeOH-CHCl3, 1:9).

¹H NMR (300 MHz, CDCl3): δ = 8.22 (s, 1 H), 7.68 (s, 1 H), 7.62 (s, 2 H), 7.19-7.43 (m, 9 H), 6.82 (d, J = 7.7 Hz, 4 H), 6.42-6.58 (m, 2 H), 5.32-5.45 (m, 1 H), 5.04 (d, J = 5.0 Hz, 1 H), 4.68-4.81 (m, 1 H), 4.38-4.46 (m, 1 H), 3.77 (s, 6 H), 3.60-3.72 (m, 1 H), 3.32-3.43 (m, 1 H), 2.92-2.99 (m, 1 H), 2.59-2.83 (m, 2 H), 2.42-2.53 (m, 1 H), 1.83 (s, 3 H), 1.55 (s, 3 H).

¹³C NMR (75 MHz, CDCl3): δ = 163.6, 163.4, 158.8, 150.5, 150.3, 146.2, 144.0, 136.7, 135.3, 135.0, 134.8, 130.0, 128.1, 127.9, 127.3, 122.3, 113.3, 111.7, 111.3, 87.2, 86.0, 85.3, 83.7, 80.1, 75.2, 62.5, 60.2, 55.2, 39.3, 38.3, 12.5, 12.0.

HRMS: m/z calcd [M + Na]+ for C42H43N7O10Na: 828.2970; found: 828.2969.

(2 R ,3 S ,5 R )-2-({1-[(2 S ,3 S ,5 R )-2-{[Bis(4-methoxyphenyl)(phenyl)methoxy]methyl}-5-[5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2 H )-yl]tetrahydrofuran-3-yl}-1 H -1,2,3-triazol-4-yl)-5-[5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2 H )-yl]tetrahydrofuran-3-yl 2-Cyanoethyl diisopropylphosphoramidite (1)

To a soln of alcohol 10 (0.1 g, 0.12 mmol) in anhyd CH2Cl2 (8 mL) at r.t. was added DIPEA (0.8 mL, 0.49 mmol) and 2-cyanoethyl diisopropylchlorophosphoramidite (0.053 g, 0.22 mmol) in CH2Cl2 (0.5 mL) under N2. After completion of the reaction (2 h), the mixture was cooled to 0 ˚C and diluted with H2O (4 mL) and CH2Cl2 (8 mL). The organic layer was separated and washed with sat. aq NaHCO3 (10 mL). The aqueous layer was extracted with CH2Cl2 (2 × 8 mL). The combined organic layers were dried (anhyd Na2SO4), filtered, and concentrated in vacuo. The residue was purified via flash chromatography (silica gel 100-200 mesh, CHCl3-MeOH-Et3N, 99:0.5:0.5) to obtain 1 (0.06 g, 49%) as a creamy powder; R f  = 0.50 (MeOH-CHCl3, 1:9).

¹H NMR (300 MHz, CDCl3): δ = 7.81 (d, J = 0.9 Hz, 1 H), 7.73 (d, J = 0.9 Hz, 1 H), 7.69 (br s, 1 H), 7.63-7.67 (m, 2 H), 7.48 (br s, 2 H), 7.36-7.42 (m, 4 H), 7.22-7.35 (m, 12 H), 6.80-6.89 (m, 8 H), 6.47-6.59 (m, 4 H), 5.28-5.40 (m, 2 H), 5.21 (br s, 1 H), 5.12 (br s, 1 H), 4.68-4.82 (m, 2 H), 4.41-4.54 (m, 2 H), 3.51-3.94 (m, 18 H), 3.30-3.39 (m, 2 H), 2.94-3.09 (m, 4 H), 2.45-2.77 (m, 8 H), 2.64 (t, J = 6.4 Hz, 4 H), 1.86 (s, 6 H), 1.56 (d, J = 5.8 Hz, 6 H), 1.24-1.10 (m, 24 H).

¹³C NMR (75 MHz, CDCl3): δ = 163.4, 163.3, 158.8, 150.3, 150.0, 146.1, 144.0, 136.6, 136.5, 135.3, 130.0, 128.1, 128.0, 127.3, 122.7, 122.2, 113.3, 111.5, 111.3, 87.2, 86.0, 85.8, 85.2, 85.0, 83.6, 83.5, 79.4, 79.1, 62.3, 62.2, 60.0, 55.2, 43.4, 43.2, 38.5, 38.4, 24.6, 24.5, 24.4, 24.4, 22.9, 12.6, 12.0.

³¹P NMR (161.91 MHz, CDCl3): δ = 150.8, 149.1.

HRMS: m/z calcd [M + Na]+ for C51H60N9O11PNa: 1028.4042; found: 1028.4048.

Acknowledgment

Ch. Nagesh thanks CSIR, New Delhi and N. Kiranmai thanks UGC, New Delhi for the award of research fellowship. The authors thank Dr. B. Jagadeesh for NMR analysis of the modified dimer nucleo­side and Dr. Ganesh Kumar for mass analysis of the oligomers.

19

Initially, the reaction was tried with n-BuLi and the yield was found to be poor as the reaction ended with an intractable mixture of products.

19

Initially, the reaction was tried with n-BuLi and the yield was found to be poor as the reaction ended with an intractable mixture of products.

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Figure 1 Some modified thymidine dinucleotides

Figure 2 Thymidine dimers

Scheme 1 Retrosynthesis for modified dimer nucleoside phosphoramidite 1

Scheme 2 Synthesis of modified dinucleotide 1 (triazole-linked dimer)

Scheme 3 Application of modified dimer in oligomer synthesis