Solid-Phase Synthesis of RNA Sequences
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89.0, 81.4, 77.0, 73.0, 67.1, 66.6, 59.4, 17.3, 17.2, 17.1, 17.0, 16.9, 16.8, 16.7,
16.6, 12.7, 12.5, 12.4, 12.3, 11.9 ppm; HRMS (ESI): m/z calcd for
C31H49N3O8SSi2: 680.2852 [M+H]+; found: 680.2854.
ed earlier in the literature.[23] These intermediates were used in the syn-
thesis of 7a–d, without purification.
2’-O-(2-Cyano-2,2-dimethylethanimine-N-oxymethyl)uridine (7a): Am-
monium fluoride (1.8 g, 50 mmol) was added to a solution of 3’,5’-O-
(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-2’-O-phthalimidooxymethyl)uri-
dine (6a, 3.3 g, 5.0 mmol) in MeOH (25 mL). The heterogeneous reaction
mixture was stirred at 258C until desilylation and dephthalimidation
were complete (16 h) as indicated by TLC (CHCl3/MeOH (9:1 v/v)). A
solution (20 mL) of 2-cyano-2-methyl propanal (2, 2.9 g, 30 mmol) in
MeOH/H2O (1:1, v/v) containing concentrated HCl (0.5 mL) was then
added to this mixture, which was stirred over 1 h at 558C. Approximately
one half of the volatiles were removed by rotoevaporation under
vacuum; the remaining material was mixed with dry silica gel (20 g) and
allowed to dry overnight. The gel mix was re-suspended in CH2Cl2
(30 mL) and layered on the top of a chromatography column packed
with silica gel (100 g) pre-equilibrated in CH2Cl2. The product was eluted
from the column using a gradient of MeOH (0!6%) in CH2Cl2. Frac-
tions containing the pure product were collected and rotoevaporated
under reduced pressure to give 7a (1.5 g, 4.1 mmol) as white solid in
82% yield. 1H NMR (300 MHz, [D6]DMSO): d=11.34 (s, 1H), 7.86 (d,
J=8.1 Hz, 1H), 7.53 (s, 1H), 5.90 (d, J=5.8 Hz, 1H), 5.64 (d, J=8.1 Hz,
1H), 5.28 (d, J=5.5 Hz, 1H), 5.16 (m, 3H), 4.26 (t, J=5.5 Hz, 1H), 4.14
(dt, J=4.6, 4.2 Hz, 1H), 3.88 (dt, J=3.6, 3.2 Hz, 1H), 3.58 (m, 2H), 1.45
(s, 3H), 1.44 ppm (s, 3H); 13C NMR (75 MHz, [D6]DMSO): d=163.2*,
163.1, 152.8, 150.7, 150.6*, 149.9*, 140.8*, 140.7, 121.8*, 121.6, 102.1,
102.0*, 96.3, 96.2* 86.4*, 86.0, 85.3, 85.2*, 79.3, 78.8*, 68.9, 68.7*, 60.9,
60.8*, 33.6, 30.8*, 24.8*, 24.7, 24.6, 24.5* ppm (signals with an asterisk
correspond to those of the minor (10–15%) oximic geometrical isomer);
HRMS (ESI): m/z calcd for C15H20N4O7: 369.1405 [M+H]+; found:
369.1405.
N6-Phenoxyacetyl-3’,5’-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-2’-O-
(methylthiomethyl)adenosine (4c): Glacial AcOH (31 mL) and Ac2O
(20 mL) was added to a solution of N6-phenoxyacetyl-5’-O-(1,1,3,3-tetrai-
sopropyldisiloxane-1,3-diyl)adenosine (12.8 g, 20.0 mmol) in DMSO
(31 mL). The solution was stirred at 508C until completion of the reac-
tion (16 h), which was monitored by TLC (CHCl3/MeOH (95:5 v/v)). The
solution was transferred to an Erlenmeyer flask (2 L) and a solution of
K2CO3 (61.6 g) in H2O (306 mL) was added, under vigorous stirring. The
precipitated material was worked-up and purified under conditions iden-
tical to those employed in the preparation of 4b. The product was isolat-
ed as
a
white solid (12.4 g, 17.6 mmol, 88%). 1H NMR (300 MHz,
[D6]DMSO): d=10.99 (s, 1H), 8.60 (s, 1H), 8.54 (s, 1H), 7.31 (m, 2H),
6.96 (m, 3H), 6.14 (s, 1H), 5.04 (s, 2H), 5.00 (m, 1H), 4.95 (d, J=3.4 Hz,
2H), 4.89 (d, J=5.2 Hz, 1H), 4.06 (m, 2H), 3.95 (d, J=12.4 Hz, 1H),
2.09 (s, 3H), 1.07–0.96 ppm (m, 28H). 13C NMR (75 MHz, [D6]DMSO):
d=167.4, 157.8, 151.6, 150.8, 149.1, 142.9, 129.5, 123.5, 121.1, 114.6, 87.7,
81.0, 76.9, 73.8, 69.4, 67.2, 60.1, 17.3, 17.17, 17.15, 17.1, 17.0, 16.97, 16.91,
16.8, 12.9, 12.8, 12.4, 12.2, 12.1 ppm; HRMS (ESI): m/z calcd for
C32H49N5O7SSi2: 704.2964 [M+H]+; found: 704.2943.
N4-Phenoxyacetyl-3’,5’-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-2’-O-
(phthalimidooxymethyl)cytidine (6b): Sulfuryl chloride (900 mL,
11.0 mmol) was added to a stirred solution of thoroughly dried 4b (6.8 g,
10 mmol) in anhydrous CH2Cl2 (100 mL). After 2 h at 258C, the solution
was concentrated under reduced pressure to give 5b as a foamy material.
N-Hydroxyphthalimide (6.5 g, 40 mmol) was placed into a separate flask
and anhydrous CH2Cl2 (50 mL) and DBU (5.2 mL, 35 mmol) was added.
After 10 min, the red solution was added to the unpurified chloromethyl
ether 5b; the reaction mixture was stirred at 258C for 24 h, at which
point CH2Cl2 (100 mL) was added. The solution was vigorously mixed
with 1m AcOH (100 mL); the aqueous layer was discarded and the or-
ganic phase was washed with a saturated aqueous solution of NaHCO3
(2ꢂ100 mL). The organic layer was dried over anhydrous Na2SO4 and fil-
tered; the filtrate was then evaporated to a foamy material under low
pressure. The crude product was purified by chromatography on silica gel
using a gradient of MeOH (0!3%) in CH2Cl2 as the eluent. Fractions
containing the pure product were collected and evaporated under
vacuum to give a yellowish solid (7.3 g, 9.2 mmol) in 92% yield based on
the molar amount of starting material (compound 4b) that was em-
ployed. 1H NMR (300 MHz, [D6]DMSO): d=11.11 (s, 1H), 8.15 (d, J=
7.6 Hz, 1H), 7.85 (m, 4H), 7.31 (t, J=7.8 Hz, 2H), 7.18 (d, J=7.6 Hz,
1H), 6.95 (m, 3H), 5.79 (s, 1H), 5.51 (dd, J=10.6, 7.0 Hz, 2H), 4.85 (s,
2H), 4.74 (d, J=4.2, Hz, 1H), 4.45 (dd, J=4.8, 4.4 Hz, 1H), 4.17 (d, J=
13.0 Hz, 1H), 4.01 (d, J=9.4 Hz, 1H), 3.93 (d, J=13.0 Hz, 1H), 1.07–
0.92 ppm (m, 28H); 13C NMR (75 MHz, [D6]DMSO): d=169.2, 163.0,
162.4, 157.7, 153.9, 145.7, 134.8, 129.5, 128.6, 123.3, 121.2, 114.4, 98.2,
95.3, 90.3, 81.0, 79.4, 68.5, 66.6, 59.9, 17.3, 17.2, 17.1, 17.0, 16.9, 16.8, 16.7,
12.5, 12.4, 12.3, 12.0 ppm; HRMS (ESI): m/z calcd for C38H50N4O11Si2:
795.3087 [M+H]+; found: 795.3060.
2’-O-(2-Cyano-2,2-dimethylethanimine-N-oxymethyl)cytidine (7b): The
synthesis of 7b was performed under conditions and at a scale identical
to those described above for the preparation of 7a, with the exception of
the silica gel purification step, which was instead performed by using a
gradient of MeOH (0!10%) in CH2Cl2. The product 7b (1.2 g,
3.4 mmol) was obtained as
a
white solid in 67% yield. 1H NMR
(300 MHz, [D6]DMSO): d=8.31 (s, 0.1H), 7.82 (d, J=7.4 Hz, 1H), 7.55
(s, 0.9H), 7.19 (s, 2H), 5.88 (d, J=4.7 Hz, 1H), 5.71 (d, J=7.4 Hz, 1H),
5.30 (dd, J=9.8, 7.8 Hz, 0.2H), 5.20 (dd, J=17.6, 7.8 Hz, 1.8H), 5.07 (m,
2H), 4.23 (t, J=4.8 Hz, 0.1H), 4.18 (t, J=4.8 Hz, 0.9H), 4.07 (q, J=
5.4 Hz, 1H), 3.83 (m, 1H), 3.65 (m, 1H), 3.55 (m, 1H), 1.49 (s, 0.3H),
1.48 (s, 0.3H), 1.46 ppm (s, 5.4H); 13C NMR (75 MHz, [D6]DMSO): d
165.5, 155.1, 155.0*, 152.7, 149.6*, 141.4, 121.7*, 121.6, 96.1*, 96.0, 94.1,
87.5, 84.3, 79.5, 79.2*, 79.1, 68.4, 68.2*, 60.5, 33.5, 30.7*, 24.6*, 24.5,
24.4 ppm (signals with an asterisk correspond to those of the minor (10–
15%) oximic geometrical isomer); HRMS (ESI): m/z calcd for
C15H21N5O6: 368.1565 [M+H]+; found: 368.1565.
2’-O-(2-Cyano-2,2-dimethylethanimine-N-oxymethyl)adenosine (7c): The
synthesis and purification of 7c were performed under conditions and at
a scale identical to those reported for the preparation of 7a. The product
1
7c (1.1 g, 2.7 mmol) was obtained as a white solid in 54% yield. H NMR
N6-Phenoxyacetyl-3’,5’-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-2’-O-
(phthalimidooxymethyl)adenosine (6c): The preparation and purification
of 6c were performed under conditions and at a scale identical to those
described above for the preparation of 6b. The product was obtained as
a yellowish solid (6.6 g, 8.1 mmol) in 81% yield based on the molar
amount of the starting material (compound 4c) that was used. 1H NMR
(300 MHz, [D6]DMSO): d=10.98 (s, 1H), 8.65 (s, 1H), 8.49 (s, 1H), 7.84
(m, 2H), 7.76 (m, 2H), 7.33 (d, J=8.4 Hz, 1H), 7.30 (d, J=8.4 Hz, 1H),
6.99 (d, J=8.4 Hz, 3H), 6.09 (s, 1H), 5.46 (d, J=7.4 Hz, 1H), 5.38–5.29
(m, 3H), 5.06 (s, 2H), 4.03 (m, 1H), 3.94 (m, 2H), 1.07–0.97 (m,
28H) ppm; 13C NMR (75 MHz, [D6]DMSO): d=167.4, 163.1, 157.8,
151.3, 150.9, 149.1, 144.6, 134.9, 129.5, 128.3, 123.6, 123.3, 121.1, 114.6,
98.5, 87.8, 80.3, 78.0, 70.4, 67.2, 60.0, 17.2, 17.1, 17.0, 16.9, 16.8, 16.7, 12.6,
12.3, 12.2, 12.1 ppm; HRMS (ESI): m/z calcd for C39H50N6O10Si2:
819.3199 [M+H]+; found: 819.3196.
(300 MHz, [D6]DMSO): d=8.33 (s, 1H), 8.13 (s, 1H), 7.35 (s, 2H), 7.27
(s, 1H), 6.01 (d, J=6.4 Hz, 1H), 5.46 (m, 1H), 5.30 (d, J=4.8 Hz, 1H),
5.12 (q, J=8.1 Hz, 2H), 4.89 (dd, J=5.0, 4.8 Hz, 1H), 4.36 (m, 1H), 4.00
(q, J=3.1 Hz, 1H), 3.68 (m, 1H), 3.57 (m, 1H), 1.35 (s, 3H), 1.30 ppm (s,
3H); 13C NMR (75 MHz, [D6]DMSO): d=156.2, 152.5, 152.4, 148.9,
139.9, 121.4, 119.3, 96.1, 86.4, 86.2, 79.2, 78.8, 69.2, 61.6, 33.3, 24.5,
24.3 ppm; HRMS (ESI): m/z calcd for C16H21N7O5: 392.1677 [M+H]+;
found: 392.1677.
2’-O-(2-Cyano-2,2-dimethylethanimine-N-oxymethyl)guanosine (7d): The
synthesis and purification of 7d were performed under conditions and at
a scale identical to those reported for the preparation of 7b. The product
7d (1.5 g, 3.6 mmol) was obtained as
a white solid in 72% yield.
1H NMR (300 MHz, [D6]DMSO): d=10.64 (s, 1H), 7.89 (s, 1H), 7.38 (s,
1H), 6.45 (s, 2H), 5.82 (d, J=6.6 Hz, 1H), 5.22 (d, J=4.8 Hz, 1H), 5.13
(dd, J=7.8, 6.6 Hz, 2H), 5.08 (t, J=5.2 Hz, 1H), 4.66 (dd, J=6.8, 6.6 Hz,
1H), 4.27 (m, 1H), 3.90 (q, J=3.2 Hz, 1H), 3.55 (m, 2H), 1.38 (s, 3H),
1.34 ppm (s, 3H); 13C NMR (75 MHz, [D6]DMSO): d=156.7, 153.7,
Synthesis of 2’-O-(aminooxymethyl)ribonucleosides (1a–d): These com-
pounds were prepared from 6a–d (10 mmol) under the conditions report-
Chem. Eur. J. 2013, 19, 4623 – 4632
ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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