Fluorous Affinity Purification of Oligonucleotides
Anal. Calcd for C47H38N3O7F17: C, 52.28, H, 3.55; N, 3.89.
Found: C, 52.14; H, 3.84; N, 4.08.
2 dd, J ) 6.6 and 4.6 Hz), 6.86-7.95 (18 H, m), 8.25 and 8.30
(1 H, 2 d, J ) 7.5 and 7.5 Hz), 9.18 (1 H, br s); partial 13C
NMR (500 MHz, CD3CN; diastereomers) δ 21.1, 25.0, 44.1,
56.0, 59.5, 59.7, 72.4, 72.5, 86.3, 86.5, 87.6, 87.8, 114.3, 119.5,
119.6, 159.9, 163.7; 31P NMR (500 MHz, CD3CN) δ 148 8 (s).
Anal. Calcd for C56H55N5O8F17P: C, 52.55; H, 4.33; N, 5.47.
Found: C, 52.44; H,4.68; N, 5.76.
N6-Benzoyl-5′-O-[4,4′-dimethoxy-4′′-(1H,1H,2H,2H-per-
fluorodecyl)trityl]-2′-deoxyadenosine, 3′-[(2-Cyanoethyl)-
(N,N-diisopropyl)]phosphoramidite (10b). A solution of
N6-benzoyl-5′-O-[4,4′-dimethoxy-4′′-(1H,1H,2H,2H-perfluoro-
decyl)trityl]-2′-deoxyadenosine (7b, 42.1 g, 38.3 mmol) in
anhydrous dichloromethane (900 mL) was treated sequentially
with 2-cyanoethyl tetraisopropylphosphorodiamidite (15.9 mL,
15.1 g, 50.0 mmol) and 1H-tetrazole (1.07 g, 15.3 mmol) at rt.
After 4 h, the mixture was washed with 5% aqueous sodium
bicarbonate, dried over sodium sulfate, and concentrated in
vacuo at e30 °C. Chromatography of this residue on silica gel
(previously deactivated with triethylamine, elution with 1:1
then 2:1 ethyl acetate/hexanes containing 0.5% triethylamine)
gave 35.5 g (71%) of the title compound as an off-white crisp
foam. HPLC analysis (10:1 acetonitrile/0.1 M triethylammo-
nium acetate) showed two diastereomers in a ratio of 57:43
(tR ) 4.15 and 5.15 min) and a total purity of >97.5: 1H NMR
(500 MHz, CD3CN) δ 1.07-1.19 (14 H, m), 2.42 (2 H, m), 2.54
and 2.65 (2 H, 2 t, J ) 6.0 and 6.0 Hz), 2.60 (1 H, m), 2.86 (2
H, m), 3.08 (1 h, m), 3.30 (2 H, m), 3.60-3.83 (2 H, m), 3.73 (3
H, 2 s), 4.19-4.23 (1 H, m), 4.90 (1 H, m), 6.44 and 6.45 (1 H,
2 dd, J ) 5.8 and 5.8 Hz), 6.76-7.99 (17 H, m), 8.25 and 8.26
(1 H, 2 s), 8.57 (1 H, s), 9.33 (1 H, br s); partial 13C NMR (500
MHz, CD3CN; diastereomers) δ 21.1, 25.0, 44.1, 56.0, 59.6,
85.65, 84.75, 86.36, 86.59, 114.1, 119.5, 119.6, 159.7; 31P NMR
(500 MHz, CD3CN): δ 148.6, 148.7 (2 s, ratio ) 59:41). Anal.
Calcd for C57H55N7O7F17P: C, 52.50; H, 4.25; N, 7.52. Found:
C, 52.19; H,4.37; N, 7.73.
5′-O-[4,4′-Dimethoxy-4′′-(1H,1H,2H,2H-perfluorodecyl)-
trityl]-N2-isobutyryl-2′-deoxyguanosine (7d). 4,4′-Di-
methoxy-4′′-[4-(1H,1H,2H,2H)-perfluorodecyl]trityl chloride (2,
FDMT-Cl, 2.47 g, 3.44 mmol) was added in three equal
portions over 1 h to a solution of commercial N2-isobutyryl-
2′-deoxyguanosine (6d, 1.07 g, 2.99 mmol) in dry pyridine (25
mL) at rt. After 18 h, methanol (3 mL) was added. After 15
min, the mixture was concentrated in vacuo and partitioned
between ethyl acetate and brine. The organic layer was dried
(sodium sulfate) and concentrated to afford an oil, which was
purified by silica gel chromatography (50:1 then 25:1 dichloro-
methane/methanol) to give 3.11 g (96%) of the title compound
as light beige glass: 1H NMR (500 MHz, CD3CN) δ 1.07 (3 H,
d, J ) 6.7 Hz), 1.33 (3 H, d, J ) 6.8 Hz), 1.24 (1 H, m), 2.30-
2.46 (3 H, m), 2.72-2.92 (3 H, m), 3.29 and 3.34 (2 H, ABq,
JAB ) 10.1 Hz, and JAX ) 3.7 Hz, JBX ) 3.7 Hz), 3.73 (3 H, s),
(3H, s), 4.13 (1 H, m), 4.71 (1 H, m), 6.19 (1 H, dd, J ) 6.5 and
6.5 Hz), 6.74-7.38 (12 H, m), 7.83 (1 H, s, 8-H), 9.13 (1 H, br
s), 12.16 (1 H, br s); partial 13C NMR (500 MHz, CD3CN) δ
18.9, 19.0, 26.1, 32.9 (t), 55.4, 64.3, 113.4, 156.1, 158.8, 180.2.
Anal. Calcd for C45H40N5O7F17: C,49.78; H, 3.71; N, 6.45.
Found: C, 49.68; H, 4.10; N, 6.59.
5′-O-[4,4′-Dimethoxy-4′′-(1H,1H,2H,2H-perfluorodecyl)-
trityl]-N2-isobutyryl-2′-deoxyguanosine, 3′-[(2-Cyanoet-
hyl)-(N,N-diisopropyl)]phosphoramidite (8d). A solution
of 5′-O-[4,4′-dimethoxy-4′′-(1H,1H,2H,2H-perfluorodecyl)trityl]-
N2-isobutyryl-2′-deoxyguanosine (7d, 51.8 g, 47.7 mmol) in
anhydrous dichloromethane (1 L) was treated sequentially
with 2-cyanoethyl tetraisopropylphosphorodiamidite (19.7 mL,
18.7 g, 62.0 mmol) and 1H-tetrazole (1.34 g, 19.1 mmol) at rt.
After 4 h, the mixture was washed with 5% aqueous sodium
bicarbonate, dried over sodium sulfate, and concentrated in
vacuo at e30 °C. Chromatography of this residue on silica gel
(elution with 10:2:1 chloroform/hexanes/triethylamine) gave
35.6 g (58%) of the title compound as an off-white crisp foam
whose purity was found to be >99% by HPLC. Impure
fractions were combined and evaporated to give another 17 g
of material, which was rechromatographed, eluting with 5:5:1
hexanes/ethyl acetate/triethylamine followed by ethyl acetate
containing 0.5% triethylamine to afford another 15.7 g (26%;
combined yield 84%) of the title compound, again as an off-
white crisp foam. HPLC analysis (10:1 acetonitrile/0.1 M
triethylammonium acetate) showed two diastereomers in a
ratio of 48:52 (tR ) 5.67 and 6.15 min) and a total purity of
>99: 1H NMR (500 MHz, CD3CN) δ 1.06-1.17 (21 H, m),
2.41-2.65 (5 H, m), 2.84-2.88 (3 H, m), 3.31 (2H, m), 3.56-
3.78 (2 H), 3.74 (6 H, 3 s), 4.20 (1 H, m), 4.68 (1 H, m), 6.23
and 6.24 (1 H, 2 dd, J ) 6.6 and 6.6 Hz), 6.75-7.33 (12 H, m),
7.81 and 7.82 (1 H, 2 s); partial 13C NMR (500 MHz, CD3CN;
diastereomers) δ 19.2, 19.3, 19.4, 24.9, 25.0, 33.0 (t), 44.1, 56.0,
59.3, 65.0, 65.1, 85.3, 85.4, 86.9, 87.0, 114.0, 119.6, 119.7, 156.4,
159.8, 180.9, 181.0; 31P NMR (500 MHz, CD3CN) δ 148.4, 148.6
(2 s ratio ) 50:50). Anal. Calcd for C54H57N7O8F17P: C, 50.40;
H, 4.47; N, 7.62. Found: C, 50.25; H,4.79; N, 7.84.
N4-Benzoyl-5′-O-[4,4′-dimethoxy-4′′-(1H,1H,2H,2H-per-
fluorodecyl)trityl]-2′-deoxycytidine (7c). 4,4′-Dimethoxy-
4′′-[4-(1H,1H,2H,2H)-perfluorodecyl]trityl chloride (2, FDMT-
Cl, 2.97 g, 3.78 mmol) was added in three equal portions over
1 h to a solution of commercial N4-benzoyl-2′-deoxycytidine (6c,
1.09 g, 3.29 mmol) in dry pyridine (25 mL) at rt. After 18 h,
methanol (3 mL) was added. After 15 min, the mixture was
concentrated in vacuo and partitioned between ethyl acetate
and brine. The organic layer was dried (sodium sulfate) and
concentrated to afford an oil, which was purified by silica gel
chromatography (50:1 then 25:1 dichloromethane/methanol)
to give 3.41 g (96%) of the title compound as a light beige
glass: 1H NMR (500 MHz, CD3CN) δ 2.30-2.42 (3 H, m), 2.75
(1 H, ddd, J ) 13.6, 6.0 and 6.0 Hz), 2.89 (2 H, t, J ) 8.5 Hz),
3.44 and 3.51 (2 H, ABq, JAB)10.9 Hz, and JAX ) 3.0 Hz, JBX
) 3.5 Hz), 3.79 (6H, s), 4.16 (1 H, m), 4.59 (1 H, dd, J ) 10.9
and 5.6 Hz), 6.29 (1 H, t, J ) 5.4 Hz), 6.18-7.85 (18 H, m),
8.39 (1 H, d, J ) 7.5 Hz), 8.85 (1 H, br s); partial 13C NMR
(500 MHz, CD3CN) δ 26.1, 32.9, 42.3, 55.4, 96.8, 113.1, 155.5,
162.5, 165.5. Anal. Calcd for C47H38N3O7F17: C, 52.28; H, 3.55;
N, 3.89. Found: C, 52.14; H, 3.84; N, 4.08.
N4-Benzoyl-5′-O-[4,4′-dimethoxy-4′′-(1H,1H,2H,2H-per-
fluorodecyl)trityl]-2′-deoxycytidine, 3′-[(2-Cyanoethyl)-
(N,N-diisopropyl)]phosphoramidite (8c). A solution of
N4-benzoyl-5′-O-[4,4′-dimethoxy-4′′-(1H,1H,2H,2H-perfluoro-
decyl)trityl]-2′-deoxycytidine (7c, 54.7 g, 50.7 mmol) in anhy-
drous dichloromethane (1 L) was treated sequentially with
2-cyanoethyl tetraisopropylphosphorodiamidite (21.0 mL, 19.9
g, 66.1 mmol) and 1H-tetrazole (1.42 g, 20.3 mmol) at rt. After
3 h, the mixture was washed with 5% aqueous sodium
bicarbonate, dried over sodium sulfate, and concentrated in
vacuo at e30 °C. Chromatography of this residue on silica gel
(previously deactivated with triethylamine, elution with 1:2
then 1:1 ethyl acetate/hexanes containing 0.5% triethylamine)
gave 40.0 g (62%) of the title compound. HPLC analysis (15:1
acetonitrile:0.1 M triethylammonium acetate) showed two
diastereomers in a ratio of 70:30 (tR ) 5.74 and 6.35 min) and
a total purity of >98%: 1H NMR (500 MHz, CD3CN) δ 0.15-
1.18 (14 H, m), 2.40 (2 H, m), 2.38 and 2.57 (2 H, 2 m), 2.54
and 2.64 (2 H, 2 t, J ) 6.0 and 6.0 Hz), 2.92 (2 H, t, J ) 8.3
Hz), 3.41 (2 H, m), 3.58-3.70 (2 H, m), 3.76 (3 H, s), 3.77 (3
H, s), 4.14-4.16 (1 H, m), 4.65-4.67 (1 H, m), 6.11-6.17 (1 H,
Relative Rates of Detritylation of 5′-O-[4,4′-Dimethoxy-
4′′-[4-(1H,1H,2H,2H)-perfluorodecyl]trityl]thymidine (7a)
and 5′-O-(4,4′-Dimethoxytrityl)thymidine (9). The proce-
dure of Takenaka and co-workers was used.8n
Determination of λmax of the Trityl Cations. The ab-
sorption spectra of the trityl cations derived from 7a and 9
were determined as follows: A solution of 3.2 mg of 7a in 3%
trichloroacetic acid (TCA) in dichloromethane was allowed to
stand for 5 min and then diluted to 1/32 of the original
concentration with 3% TCA/dichloromethane. The absorption
spectrum showed λmax ) 504 nm (21 °C). Similarly, 1.4 mg of
J. Org. Chem, Vol. 70, No. 18, 2005 7121