A R T I C L E S
Ahlborn et al.
stirred for 16 h, taken up in CH2Cl2 (70 mL), and washed thrice each
with aqueous HCl (1 M, 70 mL), aqueous NaOH (1 M, 70 mL), and
brine (70 mL). The organic layer was dried over Na2SO4 and evaporated
in vacuo. Recrystallization from EtOH gave the title compound as a
yellow solid (548 mg, 1.9 mmol, 76%). Rf ) 0.8, CHCl3/MeOH 9:1.
1H NMR (250 MHz; CDCl3): δ (ppm) ) 2.13 (m, 5H), 3.30 (t, J )
7.4 Hz, 2H), 3.96 (m, 2H), 5.52 (bs, 1H), 7.72-8.26 (m, 9H). 13C NMR
(100.6 MHz; CDCl3): δ (ppm) ) 27.20, 29.21, 32.65, 35.64, 71.63,
79.56, 123.37, 124.82, 124.97, 125.00, 125.12, 125.90, 126.77, 127.40,
127.47, 127.49, 128.81, 130.01, 130.92, 131.44, 135.69, 172.17. HR-
FAB-MS (3-NBA) calcd for C23H19NO, 325.1467; found, 325.1469.
the latter value was employed for LNA phosphoramidites, as recom-
mended by the suppliers.
Manual Chain Extension (General Protocol A). The solid support
(cpg) and the phosphoramidite (20-30 equiv) were dried at 0.1 Torr
in a polypropylene vessel. A solution of 1-H-tetrazole (0.45 M in
CH3CN, 50-200 µL, depending on the scale) was added, and the
reaction mixture was shaken on a vortexer at low speed for 1 h at room
temperature. After washing with CH3CN and CH2Cl2, oxidizer solution
for DNA synthesis (0.5 mL) was added, followed by shaking for 15
min at room temperature. The cpg was washed with CH3CN, DMF,
MeOH, and CH2Cl2, and was dried at 0.1 Torr, followed by continuation
of the DNA synthesis or deprotection.
Deprotection and Cleavage of Oligonucleotides from cpg (Gen-
eral Protocol B). At the end of DNA syntheses, the cpg was briefly
dried (0.1 Torr) and treated with saturated aqueous NH3 (1 mL) for 16
h at room temperature or 3 h at 55 °C. The supernatant was transferred
to a new vessel, and the solid support was washed with water (2 × 0.3
mL). Excess ammonia in the combined aqueous solution was removed
with a gentle stream of compressed air. The solution was filtered and
then used directly for HPLC purification.
5′-O-(4,4′-Dimethoxytrityl)-5-(3-(4-(pyren-1-yl)butyramido)propin-
1-yl)-2′-deoxyuridine (6). 5′-O-(4,4′-Dimethoxytrityl)-5-iodo-2′-deox-
yuridine (312 mg, 0.48 mmol) was dissolved in DMF (2.5 mL). The
solution was degassed in vacuo and the flask flushed with argon. Then,
NEt3 (321 µL, 231 mg, 2.29 mmol), compound 4 (465 mg, 1.43 mmol),
tetrakis(triphenylphosphine)palladium (55 mg, 48 µmol), and copper
iodide (20 mg, 0.10 mmol) were added. In each case, the mixture was
stirred until a clear solution was obtained before the next compound
was added. After 4 h at room temperature, the mixture was diluted
with ethyl acetate (70 mL) and washed thrice with aqueous EDTA
solution (5%, 70 mL) and brine (70 mL). The organic layer was dried
over Na2SO4 and the solvent was removed in vacuo. The yellow residue
was purified by column chromatography (silica, pretreated with eluant
containing 0.5% NEt3, CH2Cl2/MeOH 93:7, Rf ) 0.29) to give the title
Detritylation of Oligonucleotides During Purification (General
Protocol C). The removal of DMT or MMT groups on the 5′-terminal
nucleotide was achieved on a C-18 cartridge (Sep-Pak Vac 3 cm3, 500
mg, Milford, MA). The cartridge was first washed with CH3CN (4 mL)
and TEAA buffer (triethylammonium acetate, 0.1 M, pH 7, 6 mL).
The cartridge was loaded with the oligonucleotide dissolved in water
or TEAA buffer. Then, TEAA buffer (2 mL), trifluoroacetic acid
solution (0.2% in H2O, 4 mL), and TEAA buffer (4 mL) were passed
through the cartridge. The oligonucleotide was eluted with CH3CN/
TEAA (3:2, v/v, 4 mL) and CH3CN/TEAA (9:1, v/v, 4 mL). Fractions
were analyzed by MALDI-TOF-MS, and those containing pure product
were combined and repeatedly lyophilized from water to remove the
buffer.
1
compound as a slightly yellow solid (375 mg, 439 µmol, 92%). H
NMR (500 MHz; CDCl3): δ (ppm) ) 1.96 (t, J ) 7.2 Hz, 2H), 2.05
- 2.13 (m, 2H), 2.19-2.56 (m, 1H), 2.41-2.48 (m, 1H), 2.70 (bs,
1H), 3.25 (dd, J ) 10.8 Hz, J ) 3.3 Hz, 1H), 3.28-3.32 (m, 2H), 3.39
(dd, J ) 10.8 Hz, J ) 2.7 Hz, 1H), 3.72 (s, 6H), 3.85-3.99 (m, 2H),
4.03-4.07 (m, 1H), 4.43-4.48 (m, 1H), 5.35-5.40 (m, 1H), 6.28 (t,
J ) 6.3 Hz, 1H), 6.76-6.81 (m, 4H), 7.16-7.20 (m, 1H), 7.22-7.27
(m, 6H), 7.33-7.37 (m, 2H), 7.82 (d, J ) 7.9 Hz, 1H), 7.95-8.01 (m,
3H), 8.06-8.10 (m, 2H), 8.12-8.18 (m, 3H), 8.26 (d, J ) 9.1 Hz,
1H), 9.11 (bs, 1H). 13C NMR (100.6 MHz; CDCl3): δ (ppm) ) 27.06,
29.94, 32.67, 35.37, 41.57, 55.27, 63.43, 72.15, 74.12, 85.74, 86.64,
87.06, 89.55, 99.56, 113.36, 123.38, 124.78, 124.90, 124.93, 125.04,
125.84, 126.70, 127.00, 127.32, 127.38, 127.46, 127.84, 128.07, 128.75,
129.90, 129.97, 130.87, 131.37, 135.33, 135.49, 135.83, 142.98, 144.51,
149.09, 158.58, 158.61, 161.73, 172.04. HR-FAB-MS (3-NBA) calcd
for C53H47N3O8, 853.3363; found, 853.3365.
AT2CapPyLNA, 5′-TMS-TAAUPyTLTLUPyTLALALUPyAAUAQ
-
DP-Lys-3′ (12). Starting from solid support 9, a manual coupling cycle
with 8 according to general protocol A was performed, followed by
automated DNA synthesis with standard phosphoramidites and 1, 10,
and 11. Next, phosphoramidite 7 was coupled manually according to
general protocol A, followed by cleavage of the oligonucleotide from
solid support according to general protocol B. Crude 12 was dissolved
in 0.1 M LiOH with 20% CH3CN and HPLC purified on a C-18 column
with a gradient of CH3CN (20-70% in 40 min; Rt ) 23 min), yield of
40%. MALDI-TOF MS m/z [M - H]- calcd for C258H273N53O108P15,
6308.5; found, 6307.5.
AT, 5′-TAATTTTTAATAAT-DP-Lys-3′ (13). Prepared on solid
support 9 by standard DNA synthesis, deprotection (general protocol
B), and HPLC purification (C-18 column) with a gradient of 0% CH3CN
for 5 min, to 20% in 55 min; Rt ) 49 min, yield of 35%. MALDI-TOF
MS m/z [M - H]- calcd for C151H197N48O90P14, 4558.1; found, 4559.1.
ATCap, 5′-TMS-TAATTTTTAATAAT-DP-Lys-3′ (14). Synthe-
sized on support 9 with phosphoramidite 7 (general protocol A),
followed by cleavage (general protocol B) and HPLC purification (C-
18 column) with a gradient of 0% CH3CN for 5 min, to 25% in 30
min; Rt ) 31 min, yield of 66%. MALDI-TOF MS m/z [M - H]-
calcd for C172H221N49O97P15, 4991.5; found, 4989.2.
AT2Cap, 5′-TMS-TAATTTTTAATAAUAQ-DP-Lys-3′ (15). Start-
ing from solid support 9 phosphoramidite 8 was coupled (general
protocol A), followed by standard DNA synthesis, coupling of
phosphoramidite 7 (general protocol A), and cleavage (general protocol
B). HPLC purification (C-18 column) with a gradient of 0% B for 5
min, to 35% in 40 min; Rt ) 32 min, yield of 45%. MALDI-TOF MS
m/z [M - H]- calcd for C187H228N50O100P15, 5240.7; found, 5240.1.
AT2CapPy, 5′-TMS-TAATTTUPyTAAUPyAAUAQ-DP-Lys-3′ (16).
Synthesized identically to 15, except that phosphoramidite 1 was also
used in manual coupling steps (general protocol A). HPLC (C-18
column) with a gradient of 0% B for 5 min, to 35% in 40 min; Rt )
5′-O-(4,4′-Dimethoxytrityl)-5-(3-[4-(pyren-1-yl)butyramido]propin-
1-yl)-2′-deoxyuridin-3′-O-yl-cyanoethyl-N,N-diisopropylphosphora-
midite (1). Compound 6 (150 mg, 0.175 mmol) was dried for 16 h at
0.1 Torr, suspended in CH3CN (900 µL), and treated with DIEA (92
µL, 68 mg, 0.526 mmol). To the stirred suspension, 2-cyanoethyl-N,N-
diisopropylchlorophosphoramidite (51 µL, 54 mg, 0.228 mmol) was
added. After 15 min at room temperature, a clear, slightly yellow
solution formed, which was diluted with CH2Cl2 (30 mL) and washed
twice with saturated aqueous NaHCO3 (30 mL) and brine (30 mL),
respectively. The organic layer was dried over Na2SO4 and concentrated
to a volume of approximately 0.5 mL. The solution was added dropwise
to cyclohexane (20 mL), leading to an off-white precipitate. After
centrifugation, the supernatant was aspired, and the solid was purified
by column chromatography (silica, pretreated with eluant containing
0.5% NEt3, elution with acetone/cyclohexane 1:1, Rf ) 0.25) to give 1
as a slightly yellow solid (175.6 mg, 0.166 mmol, 95%). 31P NMR
(202 MHz; CDCl3): δ (ppm) ) 149.3, 149.7. MALDI-TOF MS
(THAP, linear positive mode) 1050.6.
Automated DNA Chain Assembly. The assembly of the unmodified
portion of oligodeoxynucleotide was performed on 1 µmol scale,
following the protocol recommended by the manufacturer of the
synthesizer (8909 Expedite synthesizer, Perseptive Biosystems, system
software 2.01), using a polypropylene reaction chamber for DNA
synthesis (Prime Synthesis, Aston, PA). Phosphoramidites of modified
residues were coupled with elongated coupling times of 3-9 min, where
9
15230 J. AM. CHEM. SOC. VOL. 129, NO. 49, 2007