Photocaging Strategy for Functionalisation of Oligonucleotides
FULL PAPER
flask was purged with nitrogen and dichloromethane (10 mL) and trie-
thylamine (3.0 equiv) were added to yield a clear colourless solution, fol-
lowed by the addition of 2-cyanoethyl diisopropylchlorophosphoramidite
(1.5 equiv). The reaction mixture was stirred at room temperature. After
disappearance of the alcohol, the reaction was stopped. The reaction so-
lution was then diluted with dichloromethane and washed with 0.1m
NaHCO3 (2ꢂ30 mL). The organic layer was collected and dried over
Na2SO4. The solvent was removed and the residue was purified by flash
column chromatography with petroleum ether/dichloromethane contain-
ing 3% triethylamine as the eluent to give 1b, 3b, 5b, 6b, 8b as a yellow
oil.
Product 1b: Yield: 71%; 1H NMR (400 MHz, CDCl3): d=7.80 (m, 2H),
7.59 (m, 1H), 7.35 (m, 1H), 5.46 (m, 1H), 3.81 (m, 1H), 3.60 (m, 2H),
3.45 (m, 1H), 2.62–2.46 (m, 2H), 1.52 (m, 3H), 1.15–0.86 ppm (m, 12H);
13C NMR (100 MHz, CDCl3): d=146.9 (m), 140.0 (m), 133.2 (m), 128.2
(m), 127.6 (m), 123.6 (m), 117.2 (m), 67.0 (dd, J=10 Hz), 58.2 (dd, J=
20 Hz), 42.9 (m), 24.9–24.0 (m), 23.8 (m), 19.9 ppm (m); 31P NMR
(162 MHz, CDCl3): d=147.7 (s), 147.3 ppm (s).
Product 3b: Yield: 59%; 1H NMR (400 MHz, CDCl3): d=7.92 (d, 1H,
J=8 Hz), 7.61 (d, 2H, J=4 Hz), 7.40 (m, 1H), 6.22 (q, 1H, J=6 Hz),
4.75 (br, 1H), 3.82 (m, 2H), 3.58 (m, 4H), 3.12 (m, 2H), 2.63 (t, 2H),
1.60 (d, 3H, J=6 Hz), 1.58 (m, 2H), 1.47 (m, 2H), 1.34 (m, 4H),
1.17 ppm (t, 12H, J=4 Hz); 13C NMR (100 MHz, CDCl3): d=155.2,
147.5, 138.7, 133.3, 128.0, 127.0, 124.2, 117.6, 68.3, 63.3 (d, J=17 Hz), 58.1
(d, J=19 Hz), 42.9 (d, J=12 Hz), 40.8, 30.9 (d, J=7 Hz), 29.7, 26.2, 25.4,
24.4 (d, J=8 Hz), 22.1, 20.2 ppm (d, J=7 Hz); 31P NMR (162 MHz,
CDCl3) d=147.2 ppm (s).
Product 5b: Yield: 63%; 1H NMR (400 MHz, CDCl3): d=7.91 (d, 1H,
J=8 Hz), 7.61 (d, 2H, J=4 Hz), 7.42 (m, 1H), 6.30 (t, 1H, J=6 Hz),
3.85–3.75 (m, 2H), 3.66–3.53 (m, 4H), 2.62 (t, 2H, J=6 Hz), 2.32 (m,
2H), 1.64–1.34 (m, 11H), 1.16 ppm (m, 12H); 13C NMR (100 MHz,
CDCl3): d=172.3, 147.8, 138.0, 133.4, 128.3, 127.1, 117.6, 67.8, 63.4, 63.2
(d, J=17 Hz), 58.2 (d, J=19 Hz), 43.0(d, J=12 Hz), 34.2, 30.8 (d, J=
8 Hz), 25.4, 24.5 (d, J=8 Hz), 21.9, 20.3 ppm (d, J=7 Hz); 31P NMR
(162 MHz, CDCl3) d=147.3 ppm (s).
Product 6b: Yield: 54%; 1H NMR (400 MHz, CDCl3): d=7.84 (d, 1H,
J=8 Hz), 7.71 (d, 1H, J=8 Hz), 7.58 (t, 1H, J=8 Hz), 7.34 (t, 1H, J=
8 Hz), 4.56 (q, 1H, J=7 Hz), 3.80 (m, 2H), 3.58 (m, 4H), 2.62 (t, 2H, J=
6 Hz), 2.30 (m, 2H0), 1.59 (d, 3H, J=7 Hz), 1.54 (m, 2 Hz), 1.44 (m,
2 Hz), 1.28 (m, 4 Hz), 1.16 ppm (t, 12H, J=7 Hz); 13C NMR (100 MHz,
CDCl3): d=149.4, 139.2, 132.8, 129.4, 127.5, 123.6, 117.6, 63.5 (d, J=
17 Hz), 58.3 (d, J=18 Hz), 43.0 (d, J=12 Hz), 38.2, 31.6, 30.9 (d, J=
7 Hz), 29.1, 28.4, 25.4, 24.5 (t, J=8 Hz), 22.9, 20.3 ppm (d, J=7 Hz);
31P NMR (162 MHz, CDCl3): d=147.3 ppm (s).
Product 7b: Yield: 73%; 1H NMR (400 MHz, CDCl3): d=7.95 (d, 1H,
J=8 Hz), 7.64 (d, 2H, J=4 Hz), 7.43 (m, 1H), 6.40 (q, 1H, J=8 Hz),
3.80 (m, 2H), 3.58 (m, 4H), 2.77 (m, 2H), 2.61 (t, 2H, J=10 Hz), 1.65 (d,
3H, J=8 Hz), 1.57 (m, 4H), 1.34 (m, 2H), 1.15 ppm (t, 12H, J=8 Hz);
13C NMR (100 MHz, CDCl3): d=170.3, 147.3, 137.5, 133.8, 128.5, 127.1,
124.4, 117.6, 70.9, 63.4 (d, J=17 Hz), 58.2 (d, J=18 Hz), 42.9 (d, J=
12 Hz), 30.9 (d, J=7 Hz), 30.8, 29.5, 28.2, 25.3, 24.5 (t, J=8 Hz), 24.4,
22.0, 20.3 ppm (d, J=7 Hz); 31P NMR (162 MHz, CDCl3): d=147.2 ppm
(s).
Product 8b: Yield: 74%; 1H NMR (400 MHz, CDCl3): d=8.04 (d, 1H,
J=8 Hz), 7.92 (d, 1H, J=8 Hz), 7.67 (t, 1H, J=8 Hz), 7.45(t, 1H, J=
8 Hz), 5.59 (m, 1H), 4.60 (t, 1H, J=8 Hz), 3.88–3.59 (m, 7H), 2.64 (t,
2H, J=3 Hz), 2.24 (t, 0.9H, J=7 Hz), 2.12 (t, 1.1H, J=7 Hz), 1.60 (s,
1.7H), 1.48 (s, 1.3H), 1.19 ppm (dd, 12H, J=4,6 Hz); 13C NMR
(100 MHz, CDCl3): d=147.3, 137.0, 136.6, 134.0, 128.4, 127.6, 124.6,
117.5, 110.5, 110.2, 74.3, 71.3, 59.6, 58.3, 43.1, 40.9, 39.5, 24.6, 23.5,
20.3 ppm; 31P NMR (162 MHz, CDCl3): d=147.7 ppm (s).
125.1, 49.6 ppm; MS (ESI-TOF+): m/z: calcd for C8H10N2O3: 205.06;
found: 205.06 [M]+.
Triethylammonium 6-[bis(4-methoxyphenyl)ACTHNUTRGNE(NUG phenyl)methoxy]hexanoate
(10): Ethyl 6-hydroxyhexanoate (200 mL, 1.23 mmol) and triethylamine
(430 mL, 3.08 mmol) were added to a solution of DMTr-Cl (500 mg,
1.48 mmol) in dichloromethane (10 mL). After stirring at room tempera-
ture for 1 h, the reaction mixture was washed with saturated aqueous
NaHCO3 (2ꢂ30 mL). The combined organic layer was dried over
Na2SO4. The solvent was removed and the residue was then dissolved in
the mixture of NaOH aqueous solution (6 molLÀ1, 25 mL) and ethanol
(4 mL). The mixture was stirred at room temperature and the reaction
was monitored by the disappearance of the ethyl ester by TLC analysis.
After completion, the mixture was diluted with CHCl3 (50 mL) and
washed with water (3ꢂ50 mL). The organic layer was concentrated and
the residue was purified by flash column chromatography with 3:7:1
methanol/dichloromethane/triethylamine as the eluent to give 10 as
a
white solid (550 mg, 1.08 mmol, 87% yield). 1H NMR (400 MHz,
CDCl3): d=7.42–7.18 (m, 10H), 6.80 (d, 4H, J=9 Hz), 3.78 (s, 6H), 3.47
(s, 1H), 3.01 (t, 2H, J=7 Hz), 2.84 (q, 6H, J=7 Hz), 2.21 (t, 2H, J=
8 Hz), 1.60 (m, 4H, J=8 Hz), 1.38 (dd, 2H, J=7,12 Hz), 1.16 ppm (t,
3H, J=7 Hz); 13C NMR (100 MHz, CDCl3): d=179.2, 158.3, 145.5, 136.8,
130.2, 128.2, 127.6, 126.5, 113.0, 85.6, 63.6, 55.2, 45.1, 36.5, 30.1, 26.4, 26.0,
9.5 ppm.
N-[2-Hydroxy-2-(2-nitrophenyl)ethyl]-4-pentynamide (11a): 4-Pentynoic
acid (640 mg, 6.51 mmol), EDC·HCl (1.24 g, 6.47 mmol) and HOBt
(996 mg, 6.50 mmol) were dissolved in DMF (5 mL) and stirred at room
temperature for 1 h. Then 9 (988 mg, 5.40 mmol) in DMF (3 mL) was
added dropwise. The reaction mixture was stirred at room temperature
for another 3 h and was then diluted with ethyl acetate (50 mL). The
ethyl acetate solution was washed with H2O (3ꢂ50 mL) and dried over
Na2SO4. The solution was concentrated and the residue was purified by
flash column chromatography with 1:1 petroleum ether/ethyl acetate as
the eluent to give 11a as a faint yellow solid (1.13 g, 4.31 mmol, 80%
yield). 1H NMR (400 MHz, CDCl3): d=7.95 (m, 2H), 7.67
ACHTUNGTRENNUNG(m, 1H),
7.45(m, 1H, J=1, 8 Hz), 6.22 (s, 1H), 5.38 (t, 1H, J=4 Hz), 3.70 (dd,
2H, J=6 Hz). 2.55 (m, 2H), 2.46 (m, 2H), 2.02 ppm (m, 1H); 13C NMR
(100 MHz, CDCl3): d=173.8, 147.7, 137.5, 133.6, 128.8, 128.5, 124.5, 82.6,
70.8, 69.7, 47.8, 35.1, 14.9 ppm; MS (ESI-TOF+): m/z: calcd for
C13H14N2O4: 285.09; found: 285.08 [M]+.
N-{2-[Bis(4-methoxyphenyl)ACTHNUGTRNEUNG(phenyl)methoxy]-2-(2-nitrophenyl)ethyl}-4-
pentynamide (11b): N-[2-Hydroxy-2-(2-nitrophenyl)ethyl]-4-pentynamide
(11a) (92 mg, 351 mmol) and DMTr-Cl (150 mg, 443 mmol) were dissolved
in pyridine (5 mL) and the reaction mixture was stirred at room tempera-
ture overnight. The solvent was removed and the residue was purified by
flash column chromatography with 1:1 petroleum ether/ethyl acetate con-
taining 1% triethylamine as the eluent to give 11b as a colourless oil
(154 mg, 273 mmol, 78% yield). 1H NMR (400 MHz, CDCl3) d=7.77 (d,
1H, J=8 Hz), 7.66 (d, 1H, J=8 Hz), 7.49–7.18 (m, 11H), 6.68 (dd, 4H,
J=9 Hz), 5.38 (t, 1H, J=5 Hz), 3.78 (d, 6H, J=8 Hz), 2.43 (m, 2H), 2.32
(m, 2H), 1.96 ppm (t, 1H, J=2 Hz); 13C NMR (100 MHz, CDCl3) d=
170.5, 158.6, 158.5, 146.8, 145.1, 138.1, 135.6, 135.3, 132.7, 130.2, 130.1,
130.0, 127.9, 127.8, 127.3, 126.9, 123.7, 113.2, 113.0, 87.6, 82.9, 69.8, 69.2,
46.2, 45.1, 35.3, 14.6 ppm; MS (ESI-TOF+): m/z: calcd for C34H32N2O6:
587.22; found: 587.26 [M]+.
1-(2-Nitrophenyl)-2-(4-pentynamido)ethyl
(12a):
6-hydroxyhexylcarbamate
N-[2-Hydroxy-2-(2-nitrophenyl)ethyl]-4-pentynamide
(11a)
(131 mg, 500 mmol), DSC (154 mg, 600 mmol) and DMAP (74 mg,
600 mmol) were dissolved in CH3CN (7 mL) under nitrogen. The mixture
was stirred at room temperature for 30 min. The solution of 6-amino-1-
hexanol (70 mg, 600 mmol) in dichloromethane (5 mL) was then added
dropwise. After stirring at room temperature for another 10 min, the mix-
ture was concentrated and the residue was purified by flash column chro-
matography with 10:1 dichloromethane/triethylamine as the eluent to
give 12a as a yellow solid (150 mg, 370 mmol, 74% yield). 1H NMR
(400 MHz, CDCl3): d=7.93 (d, 1H, J=8 Hz), 7.63 (m, 2H), 7.42 (m,
1H), 6.65 (br, 1H), 6.19 (m,1H), 5.50 (br, 1H), 3.85 (m, 1H), 3.55 (m,
3H), 3.05 (dd, 2H, J=6,12 Hz), 2.49 (s, 1H), 2.43 (m, 2H), 2.35 (d, 2H,
J=14 Hz), 1.96 (s, 1H), 1.51–1.41 (m, 4H), 1.31–1.28 ppm (m, 4H);
2-Amino-1-(2-nitrophenyl) ethanol (9): 2-Amino-1-(2-nitrophenyl) etha-
nol (9) was synthesised according to the protocol published before with
modification[53] from o-nitroacetophenone as a faint yellow solid (40%
yield over 3 steps). 1H NMR (400 MHz, [D4]MeOH) d=7.80 (dd, 2H,
J=8,11 Hz), 7.62 (t, 1H, J=7 Hz), 7.40 (t, 1H, J=8 Hz), 5.06 (d, 1H, J=
3,8 Hz), 2.90 (dd, 1H, J=2,13 Hz), 2.64 ppm (dd, 1H, J=8,13 Hz);
13C NMR (100 MHz, [D4]MeOH) d=149.4, 139.7, 134.3, 129.5, 129.4,
Chem. Eur. J. 2012, 18, 9628 – 9637
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
9635