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before opening. The reaction was then ltered through Celite®. cautiously added to the mixture. The solution was le to stir under
The product was isolated by precipitation from cold chloroform, argon until reaction completion was conrmed by TLC (ꢃ3 hours).
washing with cold chloroform, to yield an off white solid The reaction was diluted with degassed DCM (50 ml), and the
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(748 mg, 70%). H NMR (400 MHz, (CD3)2SO) d 11.56 (s, 1H), solution was washed with degassed saturated sodium bicarbonate
8.69 (t, J ¼ 5.9 Hz, 2H), 8.32 (s, 1H), 7.18 (d, J ¼ 15.6 Hz, 1H), solution (2 ꢂ 50 ml). The organic phases were collected, dried over
6.96 (d, J ¼ 15.6 Hz, 1H), 6.30–6.21 (m, 1H), 6.16–6.09 (m, 2H), sodium sulphate, and evaporated under vacuum. The crude
5.64–5.58 (m, 1H), 5.27 (d, J ¼ 4.3 Hz, 1H), 5.16 (t, J ¼ 5.0 Hz, product was puried by ash chromatography with an eluent of
1H), 4.54 (t, J ¼ 5.8 Hz, 2H), 4.29–4.23 (m, 1H), 3.82–3.78 (m, 9 : 1 DCM : 7 N ammonia in methanol. The appropriate fractions
1H), 3.70–3.55 (m, 1H), 2.18–2.13 (m, 2H). 13C NMR (100 MHz, were combined and evaporated to give an off white solid (717 mg,
(CD3)2SO) d 166.0 (C]O), 164.8 (C]O), 161.8 (C]O), 149.4 (C] 72%).
O), 142.4 (HC]C), 132.9 (HC]C), 131.5 (HC]C), 125.8 (H2C]
1H NMR (400 MHz, CDCl3) d 8.65–8.59 (m, 1H), 7.79 (d, J ¼
C), 120.8 (HC]C), 108.9 (C), 87.6 (CH), 84.6 (CH), 69.9 (CH), 21.1 Hz, 1H), 7.43–7.40 (m, 2H), 7.33–7.27 (m, 8H), 7.23–7.18
60.9 (CH2), 43.6 (CH2), 40.0 (CH2). HRMS (ES +ve) (m/z): [M + (m, 1H), 7.07 (dd, J ¼ 15.3, 2.7 Hz, 1H), 6.87–6.83 (m, 4H), 6.44
Na]+ calcd for C16H20N4O7Na, 403.1230; found 403.1229. IR neat (dd, J ¼ 17.0, 1.1 Hz, 1H), 6.28–6.23 (m, 1H), 6.16–6.09 (m, 1H),
(cmꢀ1): 3295 (m, OH/NH), 3065 (w, CH), 1689 (s, C]O), 1650 (s, 5.69 (dd, J ¼ 10.4, 1.0 Hz, 1H), 4.73 (t, J ¼ 6.0 Hz, 2H), 4.56–4.49
C]C). Mp: 250 ꢁC (degradation).
(m, 1H), 4.28–4.22 (m, 1H), 3.87–3.52 (m, 11H), 3.38–3.29 (m,
2H), 2.72–2.59 (m, 2H), 2.46 (t, J ¼ 6.4 Hz, 1H), 2.28–2.17 (m,
1H), 1.18–1.08 (m, 12H). 13C NMR (100 MHz, CDCl3) d 167.8
(C]O), 166.6 (C]O), 162.8 (C]O), 158.6 (Ar–C), 149.1 (C]O),
144.3 (Ar–C), 142.1 (HC]C), 135.6 (Ar–C), 135.4 (Ar–C), 133.5
(HC]C), 130.1 (HC]C), 130.0 (Ar–CH), 128.4 (H2C]C), 128.1
(Ar–CH), 128.0 (Ar–CH), 127.1 (Ar–CH), 122.0 (HC]C), 117.5/
117.4 (CN), 113.4 (Ar–CH), 109.9/109.8 (C), 86.8 (C), 86.3–86.0
(CH), 85.7 (CH), 74.0–73.5 (CH), 63.4/63.3 (CH2), 58.4/58.2
(CH2), 55.2 (OCH3), 44.6 (CH2), 43.4–43.2 (CH), 40.4 (CH2),
24.6 (CH3), 20.4–20.2 (CH2). 31P NMR (121 MHz, CDCl3) d 149.1,
148.6. HRMS (TOF-ES +ve) (m/z): [M]+ calcd for C46H56N6O10P,
883.3796; found 883.3793.
Synthesis of compound 3
Compound 2 (ref. 20) (1.01 g, 1.54 mmol) and palladium acetate
(35 mg, 0.16 mmol) were suspended in DMF (3 ml) in a 10 ml
microwave tube equipped with a small magnetic stirring bar.
Tributylamine (0.37 ml, 1.56 mmol) and N,N0-methyl-
enebisacrylamide (475 mg, 3.08 mmol) were then added. The
suspension was stirred and degassed with argon for 10 minutes.
The tube was tightly sealed and the mixture irradiated in a micro-
ꢁ
wave for 10 minutes at 100 C. Aer the irradiation period, the
reaction vessel was cooled to room temperature before opening.
The reaction was then ltered through Celite®, rinsing with DCM
(50 ml), and the ltrate washed with water (50 ml). The organic
phase was dried over magnesium sulphate and evaporated under
vacuum. The crude product was puried by ash chromatography
with an eluent of 9 : 1 DCM : 7 N ammonia in methanol. The
appropriate fractions were combined and evaporated to give an off
white solid (700 mg, 67%).
Synthesis of unmodied and Acrylamide-dT modied DNA
oligonucleotides
All oligonucleotides were synthesised using solid phase synthesis
on an Applied Biosystems ABI 394 DNA/RNA synthesiser using
commercially supplied DNA synthesis grade solvents and
reagents. Standard phosphoramidites of Pac-dA, iPr-Pac-dG, Ac-
dC, dT from Link Technologies, and Acrylamide-dT-CE phos-
phoramidite were used for ultramild synthesis. The phosphor-
amidites were dissolved in anhydrous acetonitrile to 0.1 M prior
to synthesis. Strands were synthesised at a 1 mmol scale on Syn-
Base™ CPG 1000/110 solid supports from Link Technologies.
Phosphoramidites were activated with 5-ethylthio-1H-tetrazole
(0.25 M) in acetonitrile prior to coupling and coupling times of 25
seconds were used. Then, phenoxyacetic anhydride and methyl-
imidazole were added to cap unreacted material, and iodine (0.02
M) in THF/pyridine/water (7 : 2 : 1) was added to oxidise the
phosphotriester formed. Upon sequence completion, the resins
were placed in freshly prepared 1 ml solutions of potassium
carbonate (0.05 M) in methanol and le overnight to cleave
strands from the resin and remove protecting groups. The solu-
tions were neutralised with acetic acid (6 ml) and the solvent was
removed on a Thermo Scientic speed vac. The dried powders
1H NMR (400 MHz, CDCl3) d 10.87 (s, 1H), 8.14 (s, 1H), 7.86
(s, 1H), 7.39–7.37 (m, 3H), 7.29–7.25 (m, 7H), 7.20–7.14 (m, 2H),
6.84 (d, J ¼ 8.7 Hz, 4H), 6.36–6.28 (m, 2H), 6.14–6.08 (m, 1H),
5.66 (d, J ¼ 11.1 Hz, 1H), 4.75 (t, J ¼ 6.6 Hz, 2H), 4.46 (s, 1H),
4.24–4.21 (m, 1H), 3.74 (s, 6H), 3.44–3.31 (m, 2H), 2.70–2.66 (m,
1H), 2.23–2.17 (m, 1H), 1.81 (s, 1H). 13C NMR (100 MHz, CDCl3)
d 168.1 (C]O), 166.8 (C]O), 162.2 (C]O), 158.8 (Ar–C), 149.6
(C]O), 144.4 (Ar–C), 142.2 (HC]C), 135.7 (Ar–C), 135.5 (Ar–C),
133.9 (HC]C), 130.3 (HC]C), 130.2 (Ar–CH), 130.0 (Ar–CH),
128.3 (H2C]C), 128.2 (Ar–CH), 127.2 (Ar–CH), 121.9 (HC]C),
113.5 (Ar–CH), 110.1 (C), 87.2 (C), 87.0 (CH), 86.8 (CH), 72.8
(CH), 64.0 (CH2), 55.4 (OCH3), 44.5 (CH2), 41.6 (CH2). HRMS (ES
+ve) (m/z): [M + H]+ calcd for C37H39N4O9, 683.2717; found
683.2715. IR neat (cmꢀ1): 3483 (b, OH/NH), 2921 (s, CH), 2851
(s, CH), 1607 (m, C]O).
Synthesis of Acrylamide-dT-CE phosphoramidite
Compound 3 (771 mg, 1.13 mmol) was dried via azeotroping with were redissolved in 1 ml Milli-Q water and desalted with a NAP-10
dry DCM (3 ꢂ 10 ml). The solid was then redissolved in dry DCM column from GE Healthcare to remove residual resin and
(15 ml), and the solution stirred under argon. DIPEA (0.49 ml, 2.84 potassium carbonate. The solutions were then concentrated to
mmol) was added via syringe to the solution. Then 2-cyanoethyl 1 ml and stored in the freezer for purication (see ESI for further
N,N-diisopropylchlorophosphoramidite (0.3 ml, 1.34 mmol) was details†).
This journal is © The Royal Society of Chemistry 2019
RSC Adv., 2019, 9, 31511–31516 | 31515