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Nucleoside Azide–Alkyne Cycloaddition Reactions Under Solvothermal Conditions or Using Copper Vials in a Ball Mill a
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Andrew J. Cummings , Francesco Ravalico , Kegan I. S. McColgana
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Bannon , Olga Eguaogie , P. Alain Elliott , Matthew R. Shannon , Iris a
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A. Bermejo , Angus Dwyer , Amanda B. Maginty , James Mack & Joseph S. Vyle
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School of Chemistry and Chemical Engineering, Queen's University Belfast, Belfast, UK b
Department of Chemistry, University of Cincinnati, Cincinnati, OH, USA Published online: 15 Apr 2015.
To cite this article: Andrew J. Cummings, Francesco Ravalico, Kegan I. S. McColgan-Bannon, Olga Eguaogie, P. Alain Elliott, Matthew R. Shannon, Iris A. Bermejo, Angus Dwyer, Amanda B. Maginty, James Mack & Joseph S. Vyle (2015) Nucleoside Azide–Alkyne Cycloaddition Reactions Under Solvothermal Conditions or Using Copper Vials in a Ball Mill, Nucleosides, Nucleotides and Nucleic Acids, 34:5, 361-370, DOI: 10.1080/15257770.2014.1001855 To link to this article: http://dx.doi.org/10.1080/15257770.2014.1001855
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Nucleosides, Nucleotides and Nucleic Acids, 34:361–370, 2015 C Taylor and Francis Group, LLC Copyright ISSN: 1525-7770 print / 1532-2335 online DOI: 10.1080/15257770.2014.1001855
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NUCLEOSIDE AZIDE–ALKYNE CYCLOADDITION REACTIONS UNDER SOLVOTHERMAL CONDITIONS OR USING COPPER VIALS IN A BALL MILL
Andrew J. Cummings,1,† Francesco Ravalico,1,2,† Kegan I. S. McColgan-Bannon,1 Olga Eguaogie,1 P. Alain Elliott,1 Matthew R. Shannon,1 Iris A. Bermejo,1 Angus Dwyer,1 Amanda B. Maginty,1 James Mack,2 and Joseph S. Vyle1 1 School of Chemistry and Chemical Engineering, Queen’s University Belfast, Belfast, UK 2 Department of Chemistry, University of Cincinnati, Cincinnati, OH, USA 2
Novel nucleoside analogues containing photoswitchable moieties were prepared using ‘click’ cycloaddition reactions between 5 -azido-5 -deoxythymidine and mono- or bis-N-propargylamidesubstituted azobenzenes. In solution, high to quantitative yields were achieved using 5 mol% Cu(I) in the presence of a stabilizing ligand. ‘Click’ reactions using the monopropargylamides were also effected in the absence of added cuprous salts by the application of liquid assisted grinding (LAG) in metallic copper reaction vials. Specifically, high speed vibration ball milling (HSVBM) using a 3/32 (2.38 mm) diameter copper ball (62 mg) at 60 Hz overnight in the presence of ethyl acetate lead to complete consumption of the 5 -azido nucleoside with clean conversion to the corresponding 1,3-triazole. Keywords
Synthetic methodology; green chemistry; modified nucleosides
INTRODUCTION Huisgen 1,3-dipolar cycloaddition reactions, in particular between organic azides and alkynes, have found increasing utility for labeling biomolecules following the discovery that rapid and regioselective reactivity could be induced under mild conditions in the presence of copper (I).[1,2] Such copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC or ‘click’) reactions have been extensively applied using nucleoside and nucleic acid derivatives typically following in situ reduction of Cu (II) in the presence of a stabilizing ligand.[3,4] ‘Click’ reactions have enabled the attachment Received 29 September 2014; accepted 19 December 2014. †These authors contributed equally to this work. Address correspondence to Joseph S. Vyle, School of Chemistry and Chemical Engineering, Queen’s University Belfast, David Keir Building, Stranmillis Road, Belfast, UK. E-mail: [email protected]
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of either alkyne or azide-derivatized nucleic acids to a variety of substrates including fluorophores, chromophores, carbohydrates, peptides, array surfaces, and other nucleic acids.[3–6] In their original report, Sharpless and coworkers described the capacity of coiled copper metal turnings (ca. 0.8 mol equivalents) to promote regioselective 1,4-triazole formation. However, compared with the growth in published examples of Cu(I)-catalyzed ‘click’ reactions in solution, relatively limited attention has been given to developing heterogeneous catalysts involving Cu(0). Examples have included the synthesis of dendrimers in the presence of copper granules[7] and several model reactions using copper nanoparticles deposited on a variety of substrates following reduction of Cu(II).[8–10] Rate enhancements for the reactions were induced using heat or microwave / ultrasonic radiation. Mechanochemistry enables both the activation of reactions in the absence of radiation and the mixing of solids with disparate solubility properties.[11,12] Although well established in materials science, only in the last two decades has the application of mechanochemistry to organic synthesis[12–14] been more rigorously explored. In particular, the synthesis of nucleoside and nucleotide derivatives using ball milling[15–18] remains undeveloped but provides an attractive target due to the ubiquity of toxic, high boiling solvents such as DMF, DMSO, or pyridine used under conventional solvothermal reaction conditions. Here we compare solution–phase conditions and vibration ball milling in copper metal vials to effect the preparation of novel triazole-linked photoswitchable nucleosides.
RESULTS AND DISCUSSION CuAAC Reactions performed upon 5 -azido-5 -deoxynucleosides have given rise to products which have been investigated as antiviral agents,[19] gelators[20], for radiolabelling[21] and bioisosteric replacements for phosphates and pyrophosphates.[22–24] Few reports appear of photoswitch attachment using ‘click’ chemistry and these are limited to reaction via 2 -Opropargyl nucleosides.[25,26] 5 -azido-5 -deoxythymidine (1)[27] was employed as the nucleoside substrate and mono N -propargyl phenylazobenzamides (2a, b or c)[16] as the alkyne components for optimizing ‘click’ reaction conditions (Scheme). Reproducible and high yields of the cycloadducts (3a–c) derived from mono-substituted propargylamides were achieved using standard solution-phase conditions in DMF. Thus, following addition of 5 mol% copper (I) bromide in the presence of a stabilizing ligand, tris(benzyltriazolylmethyl)amine (TBTA), complete consumption of the azidonucleoside (1) over 24 hours was observed. Precipitation from
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SCHEME Reagents and conditions. A: 2a–c (1.2 eq.), CuBr (5 mol%), TBTA (10 mol%), Et3 N (2.4 eq.), DMF, RT, 24 hours; or 2d (0.42 eq. to alkyne), CuBr (5 mol%), TBTA (10 mol%), Et3 N (4.8 eq.), DMF, RT, 24 hours. B: 2a–b (1.2 eq.), CuI (30 mol%), Et3 N (0.3 eq.), EtOH:MeCN:DMF (66:22:1), RT, 24 hours; C: 2a–c (1.2 eq.); EtOAc (30 eq.), 2.38 mm (3/32) diameter copper ball (62 mg), HSVBM at 60 Hz, 18 hours.
a vigorously-stirred mixture of water and ethyl acetate under aerobic conditions enabled the pure products to be isolated in very good to excellent yields (87%–93%) following filtration. The addition of EDTA was required to reduce the levels of copper (II) from the ortho-isomer (3a), although it was detected in all samples prepared
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under solution–phase conditions using 1-(2-pyridylazo)naphthol (PAN) and quantified following titration with EDTA.[28] Initial studies using the bis-propargylamide substrate (2d) in the presence of excess azidonucleoside were performed under more dilute conditions and 3 days were required to effect complete consumption of 2d. Furthermore, precipitation of the product 3d as described above resulted in the formation of a gel in the organic phase. Under saturating concentrations, only 24 hours was required to give complete reaction and by omitting ethyl acetate from the precipitation mixture, essentially quantitative recovery of the bis-clicked product was achieved. During concurrent studies using solvent-stabilized copper (I),[29] it was noted that reproducibly high yields (80–90%) of the meta-clicked product (3b) (on scales up to 1 mmol) were achieved if 30 mol% cuprous iodide was added to a solid mixture of the substrate and reactant (2b) followed by triethylamine (30 mol%) and finally solvent (3:1 ethanol:MeCN). The product was observed to precipitate shortly after commencement of stirring despite subsequent addition of DMF. Similarly, a high yield of the para-isomer (3c) was achieved under these conditions although the corresponding ortho-product (3a) could not be isolated without significant copper(II) contamination. Previous work within the Mack laboratory using high speed vibration ball milling (HSVBM) in a copper vial with a 3/16 (4.76 mm) diameter copper ball (496 mg), demonstrated that a model ‘click’ reaction using liquid reactants was complete within 15 minutes.[30] In contrast, in the current study a smaller ball (3/32 (2.38 mm) diameter; 62 mg) was employed and no reaction was observed using the dry, solid reactants. We have previously described how liquid-assisted grinding (LAG) can engender improved reaction selectivity and conversion of nucleoside and nucleotide substrates using mechanochemical activation[18] and this was also observed following addition of ethyl acetate (30 eq.) to a mixture of the azidonucleoside (1) and propargylamide (2a-c.–1.2 eq.). Complete consumption of 1 was observed following HSVBM at 60 Hz for 18 hours (the addition of smaller volumes of ethyl acetate, lower vibration frequencies, shorter reaction times or the use of noncopper balls all resulted in incomplete conversion). Pure product was isolated following trituration with dichloromethane and then refluxing ethanol. Mack reported less than 5 mg/g copper contamination by ICP-MS; no cupric ion contamination was detected in the nucleoside products using PAN. In attempting to accelerate the rate of the mechanochemicallyactivated reaction, we prepared copper vials with a larger internal volume (24.5 ml compared with 5 ml) and employed a zirconia ball (15 mm diameter, 10.69 g). LAG of 1 in the presence of one equivalent of pphenylazobenzamide (2c) and ethyl acetate (2.5 eq.) performed at 25 Hz for 40 minutes gave complete and clean conversion to the corresponding clicked product 3c (by tlc and 1H NMR). However, the physical integrity
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of the interior of the copper vial was compromised under these conditions and although attempts were made to optimize conditions using lower frequencies, the product could not be effectively isolated in the absence of contaminating fine metal powder.
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CONCLUSIONS Novel nucleoside analogues bearing photoswitchable moieties have been prepared using both solution-phase and LAG conditions. The efficiency of nucleoside or nucleotide labeling reactions such as CuAAC is often compromised by the disparate solubility profiles of the (typically hydrophobic) reactive label, the catalyst and the nucleoside or nucleotide analogue substrate. Such reactions would therefore appear to be attractive targets for mixing and activation using mechanochemistry. In this study, we have compared the efficiency of CuAAC reactions in solution and using LAG in copper vials. In the latter case, under low energy impact conditions, the click reaction can be taken to completion in the absence of cupric ion contamination following prolonged vibration in a copper vial at high frequencies. In contrast, more rapid reaction can be induced using higher energy impacts at the cost of the physical integrity of the copper surface. EXPERIMENTAL General Methods Triethylamine was refluxed and distilled from CaH2 immediately prior to use and 18.2 M water was prepared by reverse osmosis. All other reagents or solvents were purchased from commercial suppliers (Sigma, Aldrich, Fluka or Tokyo Chemical Industry UK Ltd.) and used without further purification. NMRs were recorded on Bruker Avance 400, DPX 400 or DRX 500. All spectra were recorded at ambient temperature in D6 -DMSO and chemical shifts are quoted relative to TMS. Electrospray (MS-ES+) mass spectrometry was performed using a Waters Q-TOF. HSVBM reactions were performed in a Spex Certiprep 8000D mixer/mill according to the conditions described below. 4,4 -(1,2-diazenediyl)bis[N-propyn-1-yl]-benzamide (2d) To a stirred solution of azobenzene-4,4 -dicarboxylic acid chloride[31] (0.684 g, 2.2 mmol), in anhydrous DMF (15 ml) at room temperature was added anhydrous triethylamine (0.93 ml, 6.7 mmol, 3 eq.) and propargylamine (0.37 ml, 5.8 mmol, 2.6 eq.). The reaction mixture was stirred under inert conditions for 2 hours and precipitated by dropwise addition to vigorously-stirred distilled water (155 ml). The orange solids were collected
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by filtration through a G4-sintered funnel and dried in vacuo. Yield = 0.60 g, 1.7 mmol 78%. 1 H NMR (400 MHz, D6 -DMSO) δ H = 9.16 (2H, t 3JHH = 5.5Hz, NH), 8.10 (4H, d, 3JHH = 8.7Hz, ArH), 8.01 (4H, d 3JHH = 8.7Hz, ArH), 4.11 (4H, dd, 3 JHH = 5.5Hz, 4JHH = 2.5Hz, CH2 ), 3.17 (2H, t, 4JHH = 2.5Hz, CH); 13C NMR (101MHz, D6 -DMSO) δ C = 165.11, 153.34, 136.30, 128.66, 122.61, 81.09, 73.00, 28.62. ES+MS (C20 H17 N4 O2 ), m/z = 345 (M+H+). CuAAC Reaction general procedure A. To a stirred, argon-purged solution of 5 -azido-5 -deoxythymidine (1; 170 mg, 0.64 mmol), N (phenylazo)benzamido-propyne (2a–c; 200 mg, 0.76 mmol, 1.2 eq.) and triethylamine (210 μl 1.5 mmol, 2.4eq.) in DMF (2 ml) at room temperature in the absence of light was added a freshly-prepared solution of copper (I) bromide (4.6 mg, 32 μmol, 5 mol%) and tris[(1-benzyl-1H -1,2,3triazol-4-yl)methyl]amine (34 mg, 64 μmol, 10 mol%) in argon-purged DMF (0.72 ml). These conditions were maintained for 24 hours following which the reaction mixture was added dropwise to a vigorously-stirred mixture of 1:1 water:ethyl acetate (90 ml). For the ortho-isomer EDTA.Na2 ·2H2 O (90 mg, 0.24 mmol) was added, the suspension further stirred at room temperature for 1 hour and stored at 4◦ C overnight. The pure product was isolated following filtration and washed with water and ethyl acetate and dried in vacuo. CuAAC Reaction general procedure B. To a solid mixture, at room temperature, of 5 -azido-5 -deoxythymidine (1; 53 mg, 0.20 mmol), N (phenylazo)benzamidopropyne (2a–b; 63 mg, 0.24 mmol, 1.2 eq.) and copper(I) iodide (12 mg, 63 μmol 0.3 eq.) was sequentially added triethylamine (9 μl, 65 μmol, 0.3 eq.), 3:1 ethanol:acetonitrile (7.5 ml–argon purged) under an inert atmosphere, in the absence of light. The suspension was stirred and subsequently, argon-purged DMF (1 ml) was added. These conditions were maintained for 24 hours following which the reaction mixture was diluted with ethyl acetate (50 ml). The resulting suspension was washed with saturated sodium bicarbonate (2 × 20 ml) and brine (20 ml). The organics were reduced in vacuo and triturated with diethyl ether to remove unreacted propargylamide. The pure product was dried in vacuo. Azide–alkyne cycloaddition reaction general procedure C. A custommade 2.0 × 0.5 inch screw-capped copper vial (internal volume 5 cm3) was charged with 5 -azido-5 -deoxythymidine (1; 110 mg, 0.41 mmol), N (phenylazo)benzamidopropyne (2a–c; 130 mg, 0.49 mmol, 1.2 eq.) and ethyl acetate (1.2 ml 12 mmol,30 eq.) and a 2.38 mm (3/32 inch) diameter 102 copper (99.95%) ball (62 mg). The vial was vibrated for 18 hours at 60 Hz. The reaction mixture was removed from the jar, washed with two aliquots of DCM to remove unreacted alkyne and the resulting powder suspended in ethanol, heated at reflux and then allowed to cool to room temperature. Pure product was isolated following filtration and drying in vacuo.
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5 -[4-N-methyl-(ortho-(phenyldiazenyl)benzamido)-1,2,3-triazol-1yl]-5 -deoxythymidine (3a) Procedure A: 301 mg, 0.57 mmol, 89%; Procedure C: 139 mg, 0.26 mmol, 63%. 1 H NMR (500 MHz, D6 -DMSO) δ H = 11.28 (1H, s, N3H), 8.95 (1H, t, 3JHH = 5.3Hz, CH2 NH), 7.98 (1H, s, triazole-H), 7.68-7.76 (4H, m, ArH), 7.487.62 (5H, m, ArH), 7.36 (1H, s, H6), 6.15 (1H, ψt, 3JHH = 6.9Hz, H1 ), 5.48 (1H, d, 3JHH = 4.0Hz, OH), 4.57-4.71 (2H, m, H5 , H5 ), 4.55 (2H, d, 3JHH = 5.3Hz, CH2 NH) 4.27 (1H, br s, H3 ), 4.06 (1H, br s, H4 ), 2.06-2.20 (2H, m, H2 , H2 ), 1.77 (3H, s, CH3 );13C NMR (126 MHz, DMSO) δ C = 166.68, 163.59, 151.91, 150.36, 148.63, 144.65, 136.03, 135.85 131.73, 131.12, 130.48, 129.37 (2C), 128.98, 123.73, 122.88 (2C), 115.22, 109.82, 84.01 (2C), 70.75, 51.16, 37.87, 34.86, 12.02. ES+ MS (C26 H26 N8 O5 ), m/z = 531 (M + H+), calc. 531.2104, found 531.2105. 5 -[4-N-methyl-(meta-(phenyldiazenyl)benzamido)-1,2,3-triazol-1yl]-5 -deoxythymidine (3b) Procedure A: 311 mg, 0.59 mmol, 93%; procedure B: 86 mg, 0.16 mmol, 82%; procedure C: 168 mg, 0.32 mmol, 77%. 1 H NMR (400 MHz, D6 -DMSO) δ H = 11.26 (1H, br s, N3H), 9.31 (1H, t, 3JHH = 5.4 Hz, CH2 NH), 8.39 (1H, s, ArH), 8.06 (2H, ψt, JHH = 8.0 Hz, ArH), 8.00 (1H, s, triazole-H), 7.93 (2H, d, 3JHH = 6.4 Hz, PhH), 7.71 (1H, ψt, J = 8.0Hz, ArH), 7.63 (3H, m, PhH), 7.38 (1H, s, H6), 6.17 (1H, ψt, 3JHH = 7.2Hz, H1 ), 5.40 (1H, brs, OH), 4.55–4.72 (2H, m, H5 , H5 ), 4.55 (2H, d, 3JHH = 5.2Hz, CH2 NH), 4.28 (1H, br s, H3 ), 4.06 (1H, s, H4 ), 2.05–2.20 (2H, m, H2 , H2 ), 1.78 (3H, s, CH3 ); 13C NMR (101 MHz, D6 -DMSO) δ C = 165.40, 163.63, 151.86, 151.82, 150.41, 144.98, 136.01, 135.44, 131.85, 130.06, 129.60, 129.56 (2C), 125.30, 123.76, 122.65 (2C), 121.19, 109.87, 84.05 (2C), 70.81, 51.18, 37.87, 34.97, 12.02; ES + MS (C26 H26 N8 O5 ), m/z = 531 (M + H+), calc. 531.2104, found 531.2104. 5 -[4-N-methyl-(para-(phenyldiazenyl)benzamido)-1,2,3-triazol-1yl]-5 -deoxythymidine (3c) Procedure A: 294 mg, 0.55 mmol, 87%; Procedure B: 85 mg, 0.16 mmol, 81%; Procedure C: 175 mg, 0.28 mmol, 80%. 1 H NMR (400 MHz, D6 -DMSO) δ H = 11.32 (1H, s, N3H), 9.23 (1H, t, 3JHH = 5.2Hz, CH2 NH), 8.08 (2H, d 3JHH = 8.5 Hz, ArH), 8.00 (1H, s, triazole-H), 7.89-7.98 (4H, m, ArH, PhH), 7.51-7.73 (3H, m, PhH), 7.39 (1H, s, H6), 6.17 (1H, ψt, 3JHH = 6.8Hz, H1 ), 5.50 (1H, d, 3JHH = 3.8Hz, OH), 4.58–4.75 (2H, m, H5 , H5 ), 4.54 (2H, d, 3JHH = 4.9Hz, CH2 NH), 4.27 (1H, m, H3 ), 4.08 (1H, m, H4 ), 2.04–2.21 (2H, m, H2 , H2 ), 1.78
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(3H, s, CH3 ); 13C NMR (101 MHz, DMSO) δ C = 165.36, 163.61, 153.31, 151.91, 150.39, 144.87, 136.27, 136.02, 131.99, 129.53 (2C), 128.57 (2C), 123.76, 122.72 (2C), 122.33 (2C), 109.85, 84.05 (2C), 70.80, 51.17, 37.84, 34.94, 12.01. ES + MS (C26 H26 N8 O5 ), m/z = 531 (M + H+), calc. 531.2104, found 531.2100.
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4,4’-(1,2-diazenediyl)bis[1-[5’-deoxythymidin-5’-yl]-1H-1,2,3triazol-4-ylmethyl]-benzamide (3d) A light-protected suspension of 4,4 -(1,2-diazenediyl)bis[N -propyn-1-yl]benzamide (2d; 182 mg, 0.50 mmol) in argon-purged anhydrous DMF (10 ml) was gently heated to effect partial dissolution and allowed to cool to ambient temperature under argon. 5 -azido-5 -deoxythymidine (1; 320 mg, 1.2 mmol; 2.4 eq.) and triethylamine (336 μl 2.4 mmol, 4.8 eq.) were added and the suspension stirred during addition of a freshly-prepared solution of copper (I) bromide (3.4 mg, 24 μmol, 5 mol%) and tris[(1-benzyl-1H -1,2,3triazol-4-yl)methyl]amine (26.5 mg, 50 μmol, 10 mol%) in argon-purged DMF (0.540 ml). These conditions were maintained for 24 hours during which time, complete consumption of 1 was observed. The reaction mixture was added dropwise to vigorously-stirred distilled water (150 ml) and the resultant orange solid isolated following filtration, further washing with distilled water and then drying in vacuo Yield = 437 mg, 0.50 mmol, 99%. 1 H NMR (400 MHz, D6 -DMSO) δ H = 11.32 (2H, s, N3H), 9.24 (2H, t, 3 JHH = 4.5Hz, CH2 NH), 8.10 (4H, d, 3JHH = 8.3 Hz, AB), 8.01 (2H, s, triazoleH), 8.00 (4H, d, AB), 7.39 (2H, s, H6), 6.18 (2H, ψt, 3JHH = 6.9 Hz, H1 ), 5.49 (2H, d, 3JHH = 4.0 Hz, OH), 4.57-4.75 (4H, m, H5 , H5 ), 4.56 (4H, d, 3 JHH = 4.5Hz, CH2 NH), 4.29 (2H, br s, H3 ), 4.10 (2H, m, H4 ), 2.04–2.24 (4H, m, H2 , H2 ), 1.79 (6H, s, CH3 ); 13CNMR (101 MHz, D6 -DMSO) δ C = 165.31, 163.61, 153.27, 150.39, 136.66, 136.02, 128.62 (4C), 124.18, 123.08, 122.53 (4C), 109.85, 85.50, 85.45, 70.80, 51.18, 37.84, 34.94, 12.00. ES+ MS (C40 H42 N14 O10 ), m/z = 879 (M + H+). Copper (II) Determination To a solution of 3a, 3b, 3c, or 3d in 5:1 DMSO:AcOH (ca. 8 mg/ml to typically 3 ml) was added water (2 volumes), a solution of 1-(2pyridylazo)naphthol in methanol (0.1 volume) and finally amyl alcohol (0.67 volume). In the presence of copper(II), a purple/red color was present and determined following addition of EDTA (0.01 M) with vigorous stirring until a yellow/orange color persisted in the amyl alcohol layer. ACKNOWLEDGMENTS FR was in receipt of a travel scholarship from QUB. IAB was in receipt of funding from Banco Santander for this work. We acknowledge Conor
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McGrann for performing mass spectra, Steven Heron for drilling the larger copper vials, and Patricia Martin for the synthesis of starting materials.
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REFERENCES 1. Tornoe, C.W.; Christensen, C.; Meldal, M. Peptidotriazoles on solid phase: 1,2,3 -triazoles by regiospecific copper(I)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes to azides. J. Org. Chem. 2002, 67, 3057–3064. 2. Rostovtsev, V.V.; Green, L.G.; Fokin, V.V.; Sharpless, K.B. A stepwise Huisgen cycloaddition process: Copper(I)-catalyzed regioselective “ligation” of azides and terminal alkynes. Angew. Chem. Int. Ed. 2002, 41, 2596–2599. 3. Gramlich, P.M.E.; Wirges, C.T.; Manetto, A.; Carell, T. Postsynthetic DNA modification through the copper-catalyzed azide–alkyne cycloaddition reaction. Angew. Chem. Int. Ed. 2008, 47, 8350–8358. 4. El-Sagheer, A.H.; Brown, T. Click chemistry with DNA. Chem. Soc. Rev. 2010, 39, 1388–1405. 5. Rubner, M.M.; Achatz, D.E.; Mader, H.S.; Stolwijk, J.A.; Wegener, J.; Harms, G.S.; Wolfbeis, O.S.; Wagenknecht, H.-A. DNA “Nanolamps”: “Clicked” DNA conjugates with photon upconverting nanoparticles as highly emissive biomaterial. Chempluschem 2012, 77, 129–134. 6. Chevolot, Y.; Laurenceau, E.; Phaner-Goutorbe, M.; Monnier, V.; Souteyrand, E.; Meyer, A.; Gehin, T.; Vasseur, J.J.; Morvan, F. DNA directed immobilization glycocluster array: applications and perspectives. Curr. Opin. Chem. Biol. 2014, 18, 46–54. 7. Lee, C.Y.; Held, R.; Sharma, A.; Baral, R.; Nanah, C.; Dumas, D.; Jenkins, S.; Upadhaya, S.; Du, W.J. Copper-Granule-Catalyzed Microwave-Assisted click synthesis of polyphenol dendrimers. J. Org. Chem. 2013, 78, 11221–11228. 8. Alonso, F.; Moglie, Y.; Radivoy, G.; Yus, M. Copper nanoparticles in click chemistry: an alternative catalytic system for the cycloaddition of terminal alkynes and azides. Tetrahedron Lett. 2009, 50, 2358–2362. 9. Lipshutz, B.H.; Taft, B.R. Heterogeneous copper-in-charcoal-catalyzed click chemistry. Angew. Chem. Int. Ed. 2006, 45, 8235–8238. 10. Kappe, C.O.; Van der Eycken, E. Click chemistry under non-classical reaction conditions. Chem. Soc. Rev. 2010, 39, 1280–1290. 11. Wang, G.W. Mechanochemical organic synthesis. Chem. Soc. Rev. 2013, 42, 7668–7700. 12. Gawande, M.B.; Bonifacio, V.D.B.; Luque, R.; Branco, P.S.; Varma, R.S. Solvent-Free and CatalystsFree chemistry: A benign pathway to sustainability. Chem. Sus. Chem. 2014, 7, 24–44. 13. Rodriguez, B.; Bruckmann, A.; Rantanen, T.; Bolm, C. Solvent-free carbon-carbon bond formations in ball mills. Adv. Synth. Catal. 2007, 349, 2213–2233. 14. Kaupp, G. Organic solid-state reactions with 100% yield. Top. Curr. Chem. 2005, 254, 95–183. 15. Sikchi, S.A.; Hultin, P.G. Solventless protocol for efficient bis-N -boc protection of adenosine, cytidine, and guanosine derivatives. J. Org. Chem. 2006, 71, 5888–5891. 16. Ravalico, F.; James, S.L.; Vyle, J.S. Synthesis of nucleoside analogues in a ball mill: fast, chemoselective and high yielding acylation without undesirable solvents. Green Chem. 2011, 13, 1778–1783. 17. Giri, N.; Bowen, C.; Vyle, J.S.; James, S.L. Fast, quantitative nucleoside protection under solvent-free conditions. Green Chem. 2008, 10, 627–628. 18. Ravalico, F.; Messina, I.; Berberian, M.V.; James, S.L.; Migaud, M.E.; Vyle, J.S. Rapid synthesis of nucleotide pyrophosphate linkages in a ball mill. Org. Biomol. Chem. 2011, 9, 6496–6497. 19. Zhou, L.; Amer, A.; Korn, M.; Burda, R.; Balzarini, J.; De Clercq, E.; Kern, E.R.; Torrence, P.F. Synthesis and antiviral activities of 1,2,3-triazole functionalized thymidines: 1,3-dipolar cycloaddition for efficient regioselective diversity generation. Antivir. Chem. Chemother. 2005, 16, 375–383. 20. Godeau, G.; Barthelemy, P. Glycosyl-nucleoside lipids as low-molecular-weight gelators. Langmuir 2009, 25, 8447–8450. 21. Schulz, J.; Vimont, D.; Bordenave, T.; James, D.; Escudier, J. M.; Allard, M.; Szlosek-Pinaud, M.; Fouquet, E. Silicon-Based chemistry: An original and efficient one-step approach to F-18-nucleosides and F-18-oligonucleotides for PET imaging. Chem. Eur. J. 2011, 17, 3096–3100. 22. Chen, L.Q.; Wilson, D.J.; Xu, Y.L.; Aldrich, C.C.; Felczak, K.; Sham, Y.Y.; Pankiewicz, K.W. TriazoleLinked inhibitors of inosine monophosphate dehydrogenase from human and mycobacterium tuberculosis. J. Med. Chem. 2010, 53, 4768–4778.
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23. Swarbrick, J.M.; Graeff, R.; Garnham, C.; Thomas, M.P.; Galione, A.; Potter, B.V.L. ‘Click cyclic ADP-ribose’: A neutral second messenger mimic. Chem. Commun. 2014, 50, 2458–2461. 24. Rye, C.S.; Baell, J.B. Phosphate isosteres in medicinal chemistry. Curr. Med. Chem. 2005, 12, 3127–3141. 25. Beyer, C.; Wagenknecht, H.-A. Synthesis of DNA with spirobenzopyran as an internal covalent modification. Synlett 2010, 1371–1376. 26. Freeman, C.; Vyle, J.S.; Heaney, F. Oligo switches: photoresponsive oligonucleotide conjugates by solid-supported click chemistry. RSC Adv. 2013, 3, 1652–1655. 27. Horwitz, J.P.; Tomson, A.J.; Urbanski, J.A.; Chua, J.; Nucleosides, I. 5 -Amino-5 -deoxyuridine and 5 -Amino-5 -deoxythymidine. J Org. Chem. 1962, 27, 3045–3048. 28. Cheng, K.L.; Bray, R. H. 1-(2-Pyridylazo)-2-naphthol as a possible analytical reagent. Anal. Chem. 1955, 27, 782–785. 29. Listkowski, A.; Ing, P.; Cheaib, R.; Chambert, S.; Doutheau, A.; Queneau, Y. Carboxymethylglycoside lactones (CMGLs): structural variations on the carbohydrate moiety. Tetrahedron Asymmetry 2007, 18, 2201–2210. 30. Cook, T.L.; Walker, J.A.; Mack, J. Scratching the catalytic surface of mechanochemistry: a multicomponent CuAAC reaction using a copper reaction vial. Green Chem. 2013, 15, 617–619. 31. Tomlinson, M.L. The reduction of p-nitrobenzoic acid to Hydrazo- and Azo-benzene-4,4 -dicarboxylic acids by means of glucose. J. Chem. Soc. 1946, 756.
Nucleosides, Nucleotides and Nucleic Acids Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lncn20
Nucleoside Azide–Alkyne Cycloaddition Reactions Under Solvothermal Conditions or Using Copper Vials in a Ball Mill a
ab
Andrew J. Cummings , Francesco Ravalico , Kegan I. S. McColgana
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Bannon , Olga Eguaogie , P. Alain Elliott , Matthew R. Shannon , Iris a
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A. Bermejo , Angus Dwyer , Amanda B. Maginty , James Mack & Joseph S. Vyle
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School of Chemistry and Chemical Engineering, Queen's University Belfast, Belfast, UK b
Department of Chemistry, University of Cincinnati, Cincinnati, OH, USA Published online: 15 Apr 2015.
To cite this article: Andrew J. Cummings, Francesco Ravalico, Kegan I. S. McColgan-Bannon, Olga Eguaogie, P. Alain Elliott, Matthew R. Shannon, Iris A. Bermejo, Angus Dwyer, Amanda B. Maginty, James Mack & Joseph S. Vyle (2015) Nucleoside Azide–Alkyne Cycloaddition Reactions Under Solvothermal Conditions or Using Copper Vials in a Ball Mill, Nucleosides, Nucleotides and Nucleic Acids, 34:5, 361-370, DOI: 10.1080/15257770.2014.1001855 To link to this article: http://dx.doi.org/10.1080/15257770.2014.1001855
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Nucleosides, Nucleotides and Nucleic Acids, 34:361–370, 2015 C Taylor and Francis Group, LLC Copyright ISSN: 1525-7770 print / 1532-2335 online DOI: 10.1080/15257770.2014.1001855
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NUCLEOSIDE AZIDE–ALKYNE CYCLOADDITION REACTIONS UNDER SOLVOTHERMAL CONDITIONS OR USING COPPER VIALS IN A BALL MILL
Andrew J. Cummings,1,† Francesco Ravalico,1,2,† Kegan I. S. McColgan-Bannon,1 Olga Eguaogie,1 P. Alain Elliott,1 Matthew R. Shannon,1 Iris A. Bermejo,1 Angus Dwyer,1 Amanda B. Maginty,1 James Mack,2 and Joseph S. Vyle1 1 School of Chemistry and Chemical Engineering, Queen’s University Belfast, Belfast, UK 2 Department of Chemistry, University of Cincinnati, Cincinnati, OH, USA 2
Novel nucleoside analogues containing photoswitchable moieties were prepared using ‘click’ cycloaddition reactions between 5 -azido-5 -deoxythymidine and mono- or bis-N-propargylamidesubstituted azobenzenes. In solution, high to quantitative yields were achieved using 5 mol% Cu(I) in the presence of a stabilizing ligand. ‘Click’ reactions using the monopropargylamides were also effected in the absence of added cuprous salts by the application of liquid assisted grinding (LAG) in metallic copper reaction vials. Specifically, high speed vibration ball milling (HSVBM) using a 3/32 (2.38 mm) diameter copper ball (62 mg) at 60 Hz overnight in the presence of ethyl acetate lead to complete consumption of the 5 -azido nucleoside with clean conversion to the corresponding 1,3-triazole. Keywords
Synthetic methodology; green chemistry; modified nucleosides
INTRODUCTION Huisgen 1,3-dipolar cycloaddition reactions, in particular between organic azides and alkynes, have found increasing utility for labeling biomolecules following the discovery that rapid and regioselective reactivity could be induced under mild conditions in the presence of copper (I).[1,2] Such copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC or ‘click’) reactions have been extensively applied using nucleoside and nucleic acid derivatives typically following in situ reduction of Cu (II) in the presence of a stabilizing ligand.[3,4] ‘Click’ reactions have enabled the attachment Received 29 September 2014; accepted 19 December 2014. †These authors contributed equally to this work. Address correspondence to Joseph S. Vyle, School of Chemistry and Chemical Engineering, Queen’s University Belfast, David Keir Building, Stranmillis Road, Belfast, UK. E-mail: [email protected]
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of either alkyne or azide-derivatized nucleic acids to a variety of substrates including fluorophores, chromophores, carbohydrates, peptides, array surfaces, and other nucleic acids.[3–6] In their original report, Sharpless and coworkers described the capacity of coiled copper metal turnings (ca. 0.8 mol equivalents) to promote regioselective 1,4-triazole formation. However, compared with the growth in published examples of Cu(I)-catalyzed ‘click’ reactions in solution, relatively limited attention has been given to developing heterogeneous catalysts involving Cu(0). Examples have included the synthesis of dendrimers in the presence of copper granules[7] and several model reactions using copper nanoparticles deposited on a variety of substrates following reduction of Cu(II).[8–10] Rate enhancements for the reactions were induced using heat or microwave / ultrasonic radiation. Mechanochemistry enables both the activation of reactions in the absence of radiation and the mixing of solids with disparate solubility properties.[11,12] Although well established in materials science, only in the last two decades has the application of mechanochemistry to organic synthesis[12–14] been more rigorously explored. In particular, the synthesis of nucleoside and nucleotide derivatives using ball milling[15–18] remains undeveloped but provides an attractive target due to the ubiquity of toxic, high boiling solvents such as DMF, DMSO, or pyridine used under conventional solvothermal reaction conditions. Here we compare solution–phase conditions and vibration ball milling in copper metal vials to effect the preparation of novel triazole-linked photoswitchable nucleosides.
RESULTS AND DISCUSSION CuAAC Reactions performed upon 5 -azido-5 -deoxynucleosides have given rise to products which have been investigated as antiviral agents,[19] gelators[20], for radiolabelling[21] and bioisosteric replacements for phosphates and pyrophosphates.[22–24] Few reports appear of photoswitch attachment using ‘click’ chemistry and these are limited to reaction via 2 -Opropargyl nucleosides.[25,26] 5 -azido-5 -deoxythymidine (1)[27] was employed as the nucleoside substrate and mono N -propargyl phenylazobenzamides (2a, b or c)[16] as the alkyne components for optimizing ‘click’ reaction conditions (Scheme). Reproducible and high yields of the cycloadducts (3a–c) derived from mono-substituted propargylamides were achieved using standard solution-phase conditions in DMF. Thus, following addition of 5 mol% copper (I) bromide in the presence of a stabilizing ligand, tris(benzyltriazolylmethyl)amine (TBTA), complete consumption of the azidonucleoside (1) over 24 hours was observed. Precipitation from
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SCHEME Reagents and conditions. A: 2a–c (1.2 eq.), CuBr (5 mol%), TBTA (10 mol%), Et3 N (2.4 eq.), DMF, RT, 24 hours; or 2d (0.42 eq. to alkyne), CuBr (5 mol%), TBTA (10 mol%), Et3 N (4.8 eq.), DMF, RT, 24 hours. B: 2a–b (1.2 eq.), CuI (30 mol%), Et3 N (0.3 eq.), EtOH:MeCN:DMF (66:22:1), RT, 24 hours; C: 2a–c (1.2 eq.); EtOAc (30 eq.), 2.38 mm (3/32) diameter copper ball (62 mg), HSVBM at 60 Hz, 18 hours.
a vigorously-stirred mixture of water and ethyl acetate under aerobic conditions enabled the pure products to be isolated in very good to excellent yields (87%–93%) following filtration. The addition of EDTA was required to reduce the levels of copper (II) from the ortho-isomer (3a), although it was detected in all samples prepared
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under solution–phase conditions using 1-(2-pyridylazo)naphthol (PAN) and quantified following titration with EDTA.[28] Initial studies using the bis-propargylamide substrate (2d) in the presence of excess azidonucleoside were performed under more dilute conditions and 3 days were required to effect complete consumption of 2d. Furthermore, precipitation of the product 3d as described above resulted in the formation of a gel in the organic phase. Under saturating concentrations, only 24 hours was required to give complete reaction and by omitting ethyl acetate from the precipitation mixture, essentially quantitative recovery of the bis-clicked product was achieved. During concurrent studies using solvent-stabilized copper (I),[29] it was noted that reproducibly high yields (80–90%) of the meta-clicked product (3b) (on scales up to 1 mmol) were achieved if 30 mol% cuprous iodide was added to a solid mixture of the substrate and reactant (2b) followed by triethylamine (30 mol%) and finally solvent (3:1 ethanol:MeCN). The product was observed to precipitate shortly after commencement of stirring despite subsequent addition of DMF. Similarly, a high yield of the para-isomer (3c) was achieved under these conditions although the corresponding ortho-product (3a) could not be isolated without significant copper(II) contamination. Previous work within the Mack laboratory using high speed vibration ball milling (HSVBM) in a copper vial with a 3/16 (4.76 mm) diameter copper ball (496 mg), demonstrated that a model ‘click’ reaction using liquid reactants was complete within 15 minutes.[30] In contrast, in the current study a smaller ball (3/32 (2.38 mm) diameter; 62 mg) was employed and no reaction was observed using the dry, solid reactants. We have previously described how liquid-assisted grinding (LAG) can engender improved reaction selectivity and conversion of nucleoside and nucleotide substrates using mechanochemical activation[18] and this was also observed following addition of ethyl acetate (30 eq.) to a mixture of the azidonucleoside (1) and propargylamide (2a-c.–1.2 eq.). Complete consumption of 1 was observed following HSVBM at 60 Hz for 18 hours (the addition of smaller volumes of ethyl acetate, lower vibration frequencies, shorter reaction times or the use of noncopper balls all resulted in incomplete conversion). Pure product was isolated following trituration with dichloromethane and then refluxing ethanol. Mack reported less than 5 mg/g copper contamination by ICP-MS; no cupric ion contamination was detected in the nucleoside products using PAN. In attempting to accelerate the rate of the mechanochemicallyactivated reaction, we prepared copper vials with a larger internal volume (24.5 ml compared with 5 ml) and employed a zirconia ball (15 mm diameter, 10.69 g). LAG of 1 in the presence of one equivalent of pphenylazobenzamide (2c) and ethyl acetate (2.5 eq.) performed at 25 Hz for 40 minutes gave complete and clean conversion to the corresponding clicked product 3c (by tlc and 1H NMR). However, the physical integrity
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of the interior of the copper vial was compromised under these conditions and although attempts were made to optimize conditions using lower frequencies, the product could not be effectively isolated in the absence of contaminating fine metal powder.
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CONCLUSIONS Novel nucleoside analogues bearing photoswitchable moieties have been prepared using both solution-phase and LAG conditions. The efficiency of nucleoside or nucleotide labeling reactions such as CuAAC is often compromised by the disparate solubility profiles of the (typically hydrophobic) reactive label, the catalyst and the nucleoside or nucleotide analogue substrate. Such reactions would therefore appear to be attractive targets for mixing and activation using mechanochemistry. In this study, we have compared the efficiency of CuAAC reactions in solution and using LAG in copper vials. In the latter case, under low energy impact conditions, the click reaction can be taken to completion in the absence of cupric ion contamination following prolonged vibration in a copper vial at high frequencies. In contrast, more rapid reaction can be induced using higher energy impacts at the cost of the physical integrity of the copper surface. EXPERIMENTAL General Methods Triethylamine was refluxed and distilled from CaH2 immediately prior to use and 18.2 M water was prepared by reverse osmosis. All other reagents or solvents were purchased from commercial suppliers (Sigma, Aldrich, Fluka or Tokyo Chemical Industry UK Ltd.) and used without further purification. NMRs were recorded on Bruker Avance 400, DPX 400 or DRX 500. All spectra were recorded at ambient temperature in D6 -DMSO and chemical shifts are quoted relative to TMS. Electrospray (MS-ES+) mass spectrometry was performed using a Waters Q-TOF. HSVBM reactions were performed in a Spex Certiprep 8000D mixer/mill according to the conditions described below. 4,4 -(1,2-diazenediyl)bis[N-propyn-1-yl]-benzamide (2d) To a stirred solution of azobenzene-4,4 -dicarboxylic acid chloride[31] (0.684 g, 2.2 mmol), in anhydrous DMF (15 ml) at room temperature was added anhydrous triethylamine (0.93 ml, 6.7 mmol, 3 eq.) and propargylamine (0.37 ml, 5.8 mmol, 2.6 eq.). The reaction mixture was stirred under inert conditions for 2 hours and precipitated by dropwise addition to vigorously-stirred distilled water (155 ml). The orange solids were collected
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by filtration through a G4-sintered funnel and dried in vacuo. Yield = 0.60 g, 1.7 mmol 78%. 1 H NMR (400 MHz, D6 -DMSO) δ H = 9.16 (2H, t 3JHH = 5.5Hz, NH), 8.10 (4H, d, 3JHH = 8.7Hz, ArH), 8.01 (4H, d 3JHH = 8.7Hz, ArH), 4.11 (4H, dd, 3 JHH = 5.5Hz, 4JHH = 2.5Hz, CH2 ), 3.17 (2H, t, 4JHH = 2.5Hz, CH); 13C NMR (101MHz, D6 -DMSO) δ C = 165.11, 153.34, 136.30, 128.66, 122.61, 81.09, 73.00, 28.62. ES+MS (C20 H17 N4 O2 ), m/z = 345 (M+H+). CuAAC Reaction general procedure A. To a stirred, argon-purged solution of 5 -azido-5 -deoxythymidine (1; 170 mg, 0.64 mmol), N (phenylazo)benzamido-propyne (2a–c; 200 mg, 0.76 mmol, 1.2 eq.) and triethylamine (210 μl 1.5 mmol, 2.4eq.) in DMF (2 ml) at room temperature in the absence of light was added a freshly-prepared solution of copper (I) bromide (4.6 mg, 32 μmol, 5 mol%) and tris[(1-benzyl-1H -1,2,3triazol-4-yl)methyl]amine (34 mg, 64 μmol, 10 mol%) in argon-purged DMF (0.72 ml). These conditions were maintained for 24 hours following which the reaction mixture was added dropwise to a vigorously-stirred mixture of 1:1 water:ethyl acetate (90 ml). For the ortho-isomer EDTA.Na2 ·2H2 O (90 mg, 0.24 mmol) was added, the suspension further stirred at room temperature for 1 hour and stored at 4◦ C overnight. The pure product was isolated following filtration and washed with water and ethyl acetate and dried in vacuo. CuAAC Reaction general procedure B. To a solid mixture, at room temperature, of 5 -azido-5 -deoxythymidine (1; 53 mg, 0.20 mmol), N (phenylazo)benzamidopropyne (2a–b; 63 mg, 0.24 mmol, 1.2 eq.) and copper(I) iodide (12 mg, 63 μmol 0.3 eq.) was sequentially added triethylamine (9 μl, 65 μmol, 0.3 eq.), 3:1 ethanol:acetonitrile (7.5 ml–argon purged) under an inert atmosphere, in the absence of light. The suspension was stirred and subsequently, argon-purged DMF (1 ml) was added. These conditions were maintained for 24 hours following which the reaction mixture was diluted with ethyl acetate (50 ml). The resulting suspension was washed with saturated sodium bicarbonate (2 × 20 ml) and brine (20 ml). The organics were reduced in vacuo and triturated with diethyl ether to remove unreacted propargylamide. The pure product was dried in vacuo. Azide–alkyne cycloaddition reaction general procedure C. A custommade 2.0 × 0.5 inch screw-capped copper vial (internal volume 5 cm3) was charged with 5 -azido-5 -deoxythymidine (1; 110 mg, 0.41 mmol), N (phenylazo)benzamidopropyne (2a–c; 130 mg, 0.49 mmol, 1.2 eq.) and ethyl acetate (1.2 ml 12 mmol,30 eq.) and a 2.38 mm (3/32 inch) diameter 102 copper (99.95%) ball (62 mg). The vial was vibrated for 18 hours at 60 Hz. The reaction mixture was removed from the jar, washed with two aliquots of DCM to remove unreacted alkyne and the resulting powder suspended in ethanol, heated at reflux and then allowed to cool to room temperature. Pure product was isolated following filtration and drying in vacuo.
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5 -[4-N-methyl-(ortho-(phenyldiazenyl)benzamido)-1,2,3-triazol-1yl]-5 -deoxythymidine (3a) Procedure A: 301 mg, 0.57 mmol, 89%; Procedure C: 139 mg, 0.26 mmol, 63%. 1 H NMR (500 MHz, D6 -DMSO) δ H = 11.28 (1H, s, N3H), 8.95 (1H, t, 3JHH = 5.3Hz, CH2 NH), 7.98 (1H, s, triazole-H), 7.68-7.76 (4H, m, ArH), 7.487.62 (5H, m, ArH), 7.36 (1H, s, H6), 6.15 (1H, ψt, 3JHH = 6.9Hz, H1 ), 5.48 (1H, d, 3JHH = 4.0Hz, OH), 4.57-4.71 (2H, m, H5 , H5 ), 4.55 (2H, d, 3JHH = 5.3Hz, CH2 NH) 4.27 (1H, br s, H3 ), 4.06 (1H, br s, H4 ), 2.06-2.20 (2H, m, H2 , H2 ), 1.77 (3H, s, CH3 );13C NMR (126 MHz, DMSO) δ C = 166.68, 163.59, 151.91, 150.36, 148.63, 144.65, 136.03, 135.85 131.73, 131.12, 130.48, 129.37 (2C), 128.98, 123.73, 122.88 (2C), 115.22, 109.82, 84.01 (2C), 70.75, 51.16, 37.87, 34.86, 12.02. ES+ MS (C26 H26 N8 O5 ), m/z = 531 (M + H+), calc. 531.2104, found 531.2105. 5 -[4-N-methyl-(meta-(phenyldiazenyl)benzamido)-1,2,3-triazol-1yl]-5 -deoxythymidine (3b) Procedure A: 311 mg, 0.59 mmol, 93%; procedure B: 86 mg, 0.16 mmol, 82%; procedure C: 168 mg, 0.32 mmol, 77%. 1 H NMR (400 MHz, D6 -DMSO) δ H = 11.26 (1H, br s, N3H), 9.31 (1H, t, 3JHH = 5.4 Hz, CH2 NH), 8.39 (1H, s, ArH), 8.06 (2H, ψt, JHH = 8.0 Hz, ArH), 8.00 (1H, s, triazole-H), 7.93 (2H, d, 3JHH = 6.4 Hz, PhH), 7.71 (1H, ψt, J = 8.0Hz, ArH), 7.63 (3H, m, PhH), 7.38 (1H, s, H6), 6.17 (1H, ψt, 3JHH = 7.2Hz, H1 ), 5.40 (1H, brs, OH), 4.55–4.72 (2H, m, H5 , H5 ), 4.55 (2H, d, 3JHH = 5.2Hz, CH2 NH), 4.28 (1H, br s, H3 ), 4.06 (1H, s, H4 ), 2.05–2.20 (2H, m, H2 , H2 ), 1.78 (3H, s, CH3 ); 13C NMR (101 MHz, D6 -DMSO) δ C = 165.40, 163.63, 151.86, 151.82, 150.41, 144.98, 136.01, 135.44, 131.85, 130.06, 129.60, 129.56 (2C), 125.30, 123.76, 122.65 (2C), 121.19, 109.87, 84.05 (2C), 70.81, 51.18, 37.87, 34.97, 12.02; ES + MS (C26 H26 N8 O5 ), m/z = 531 (M + H+), calc. 531.2104, found 531.2104. 5 -[4-N-methyl-(para-(phenyldiazenyl)benzamido)-1,2,3-triazol-1yl]-5 -deoxythymidine (3c) Procedure A: 294 mg, 0.55 mmol, 87%; Procedure B: 85 mg, 0.16 mmol, 81%; Procedure C: 175 mg, 0.28 mmol, 80%. 1 H NMR (400 MHz, D6 -DMSO) δ H = 11.32 (1H, s, N3H), 9.23 (1H, t, 3JHH = 5.2Hz, CH2 NH), 8.08 (2H, d 3JHH = 8.5 Hz, ArH), 8.00 (1H, s, triazole-H), 7.89-7.98 (4H, m, ArH, PhH), 7.51-7.73 (3H, m, PhH), 7.39 (1H, s, H6), 6.17 (1H, ψt, 3JHH = 6.8Hz, H1 ), 5.50 (1H, d, 3JHH = 3.8Hz, OH), 4.58–4.75 (2H, m, H5 , H5 ), 4.54 (2H, d, 3JHH = 4.9Hz, CH2 NH), 4.27 (1H, m, H3 ), 4.08 (1H, m, H4 ), 2.04–2.21 (2H, m, H2 , H2 ), 1.78
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(3H, s, CH3 ); 13C NMR (101 MHz, DMSO) δ C = 165.36, 163.61, 153.31, 151.91, 150.39, 144.87, 136.27, 136.02, 131.99, 129.53 (2C), 128.57 (2C), 123.76, 122.72 (2C), 122.33 (2C), 109.85, 84.05 (2C), 70.80, 51.17, 37.84, 34.94, 12.01. ES + MS (C26 H26 N8 O5 ), m/z = 531 (M + H+), calc. 531.2104, found 531.2100.
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4,4’-(1,2-diazenediyl)bis[1-[5’-deoxythymidin-5’-yl]-1H-1,2,3triazol-4-ylmethyl]-benzamide (3d) A light-protected suspension of 4,4 -(1,2-diazenediyl)bis[N -propyn-1-yl]benzamide (2d; 182 mg, 0.50 mmol) in argon-purged anhydrous DMF (10 ml) was gently heated to effect partial dissolution and allowed to cool to ambient temperature under argon. 5 -azido-5 -deoxythymidine (1; 320 mg, 1.2 mmol; 2.4 eq.) and triethylamine (336 μl 2.4 mmol, 4.8 eq.) were added and the suspension stirred during addition of a freshly-prepared solution of copper (I) bromide (3.4 mg, 24 μmol, 5 mol%) and tris[(1-benzyl-1H -1,2,3triazol-4-yl)methyl]amine (26.5 mg, 50 μmol, 10 mol%) in argon-purged DMF (0.540 ml). These conditions were maintained for 24 hours during which time, complete consumption of 1 was observed. The reaction mixture was added dropwise to vigorously-stirred distilled water (150 ml) and the resultant orange solid isolated following filtration, further washing with distilled water and then drying in vacuo Yield = 437 mg, 0.50 mmol, 99%. 1 H NMR (400 MHz, D6 -DMSO) δ H = 11.32 (2H, s, N3H), 9.24 (2H, t, 3 JHH = 4.5Hz, CH2 NH), 8.10 (4H, d, 3JHH = 8.3 Hz, AB), 8.01 (2H, s, triazoleH), 8.00 (4H, d, AB), 7.39 (2H, s, H6), 6.18 (2H, ψt, 3JHH = 6.9 Hz, H1 ), 5.49 (2H, d, 3JHH = 4.0 Hz, OH), 4.57-4.75 (4H, m, H5 , H5 ), 4.56 (4H, d, 3 JHH = 4.5Hz, CH2 NH), 4.29 (2H, br s, H3 ), 4.10 (2H, m, H4 ), 2.04–2.24 (4H, m, H2 , H2 ), 1.79 (6H, s, CH3 ); 13CNMR (101 MHz, D6 -DMSO) δ C = 165.31, 163.61, 153.27, 150.39, 136.66, 136.02, 128.62 (4C), 124.18, 123.08, 122.53 (4C), 109.85, 85.50, 85.45, 70.80, 51.18, 37.84, 34.94, 12.00. ES+ MS (C40 H42 N14 O10 ), m/z = 879 (M + H+). Copper (II) Determination To a solution of 3a, 3b, 3c, or 3d in 5:1 DMSO:AcOH (ca. 8 mg/ml to typically 3 ml) was added water (2 volumes), a solution of 1-(2pyridylazo)naphthol in methanol (0.1 volume) and finally amyl alcohol (0.67 volume). In the presence of copper(II), a purple/red color was present and determined following addition of EDTA (0.01 M) with vigorous stirring until a yellow/orange color persisted in the amyl alcohol layer. ACKNOWLEDGMENTS FR was in receipt of a travel scholarship from QUB. IAB was in receipt of funding from Banco Santander for this work. We acknowledge Conor
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McGrann for performing mass spectra, Steven Heron for drilling the larger copper vials, and Patricia Martin for the synthesis of starting materials.
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