750 Bioconjugate Chem., Vol. 21, No. 4, 2010
Karskela et al.
at room temperature and evaporated to dryness. Crude product
was purified by silica gel chromatography (1% MeOH in
CH2Cl2) to yield 3.01 g (53%) of 10 as white foam. 1H NMR
(500 MHz, CDCl3): δppm 7.71 (m, 8H, Pht), 7.44 (m, 4H,
MMTr), 7.31 (m, 2H, MMTr), 7.26-7.16 (m, 7H, MMTr
and Ar), 7.12 (br d, 2H, Ar), 6.78 (m, 2H, MMTr), 5.18 (s,
4H, 2 × CH2ON), 4.19 (t, 2H, J ) 6.2 Hz, CH2OAr), 3.76
(s, 3H, CH3), 3.27 (t, 2H, J ) 6.0 Hz, MMTrOCH2), 2.06
(tt, 2H, J ) 6.0 and 6.2 Hz, CH2CH2CH2); 13C NMR (125
MHz, CDCl3): δppm 163.4, 159.2, 158.4, 144.7, 136.0, 135.4,
134.4, 130.3, 128.9, 128.4, 127.7, 126.7, 123.5, 122.7, 116.5,
113.0, 86.2, 79.4, 65.0, 59.8, 55.2, 29.9; HRMS (ESI) [M +
Na]+ C47H38N2NaO9 requires 797.2475, found 797.2512.
silica gel chromatography (0.1% triethylamine and 3% MeOH
in CH2Cl2) to yield 1.90 g (93%) of 14. H NMR (500 MHz,
1
CDCl3): δppm 7.82 (m, 2H, Pht), 7.73 (m, 2H, Pht), 7.44 (m,
2H, DMTr), 7.33 (m, 4H, DMTr), 7.27 (m, 2H, DMTr), 7.18
(m, 1H, DMTr), 6.82 (m, 4H, DMTr), 4.40-4.31 (m, 3H,
CH2ON and H-1), 4.25 (m, 1H, H-3), 3.88 (m, 1H, H-4), 3.79
(s, 6H, 2 × OCH3), 3.07 (m, 2H, H-5), 2.08 (m, 2H,
CH2CH2ON), 1.94 (m, 1H, H-2a), 1.74 (m, 1H, H-2b), 0.84 [s,
9H, C(CH3)3], 0.01 (s, 3H, CH3Si), -0.02 (s, 3H, CH3Si); 13C
NMR (125 MHz, CDCl3): δppm 163.6, 158.4, 145.0, 136.2,
134.4, 130.1, 128.9, 128.2, 127.7, 126.6, 123.5, 113.0, 86.4,
85.9, 76.1, 75.2, 74.3, 64.3, 55.2, 41.3, 34.2, 25.8, 18.0, -4.6,
-4.8; HRMS (ESI) [M + Na]+ C42H49NNaO8Si requires
746.3125, found 746.3147.
3-[3,5-Bis(phthalimidoxymethyl)phenoxy]propanol (11). Com-
pound 10 (3.01 g, 3.88 mmol) was suspended in 80% aqueous
acetic acid (40 mL) and stirred for 4.5 h. The mixture was
evaporated to dryness, and the residue was dissolved in
CH2Cl2 and washed with saturated NaHCO3. The organic
layer was separated and the aqueous layer was extracted four
times with CH2Cl2. The combined organic layers were dried
with Na2SO4 and evaporated to dryness. The residue was
purified by silica gel chromatography (4-5% MeOH in
5-(4,4′-Dimethoxytrityl)-1,2-dideoxy-1-C-(2-phthalimidoxy-
ethyl)-ꢀ-D-erythro-pentofuranose (15). Triethylamine trihy-
drofluoride (409 µL, 2.51 mmol) was added to a solution of
14 (1.21 g, 1.67 mmol) in dry THF (15 mL). The mixture
was stirred at room temperature for 24 h, after which another
portion of triethylamine trihydrofluoride (408 µL, 2.50 mmol)
was added. The reaction was allowed to proceed for another
24 h, followed by addition of triethylamine (200 µL, 1.43
mmol) and evaporation. The crude product was purified by
silica gel chromatography (0.1% triethylamine and 3% MeOH
in CH2Cl2) to yield 0.538 g (53%) of 15. 1H NMR (500 MHz,
CDCl3): δppm 7.82 (m, 2H, Pht), 7.73 (m, 2H, Pht), 7.42 (m,
2H, DMTr), 7.32 (m, 4H, DMTr), 7.27 (m, 2H, DMTr), 7.19
(m, 1H, DMTr), 6.82 (m, 4H, DMTr), 4.42-4.29 (m, 4H,
CH2ON, H-1, and H-3), 3.91 (m, 1H, H-4), 3.79 (s, 6H, 2 ×
OCH3), 3.23 (dd, 1H, J ) 4.6 and 9.6 Hz, H-5a), 3.08 (dd,
1H, J ) 5.9 and 9.6 Hz, H-5b), 2.10-2.05 (m, 3H,
CH2CH2ON and H-2a), 1.90-1.84 (m, 2H, H-2b and OH);
13C NMR (125 MHz, CDCl3): δppm 163.6, 158.5, 144.9, 136.1,
134.5, 130.1, 128.9, 128.2, 127.8, 126.8, 123.5, 113.1, 86.1,
85.8, 75.9, 75.3, 74.7, 64.7, 55.2, 40.9, 34.2; HRMS (ESI):
[M + Na]+ C36H35NNaO8 requires 632.2261, found 632.2268.
5-(4,4′-Dimethoxytrityl)-1,2-dideoxy-1-C-(2-phthalimidoxy-
ethyl)-ꢀ-D-erythro-pentofuranosyl-3-(2-cyanoethyl-N,N-diiso-
propylphosphoramidite) (16). 2-Cyanoethyl N,N,N′,N’-tet-
raisopropylphosphordiamidite (365 µL, 1.15 mmol) was
added to a solution of 15 (0.538 g, 0.884 mmol) in dry MeCN
(1 mL), followed by addition of tetrazole as 0.45 mol L-1
solution in MeCN (2.0 mL, 0.90 mmol). After 80 min, the
mixture was evaporated to dryness, saturated NaHCO3 was
added, and the product was extracted twice with CH2Cl2. The
combined organic layers were dried with Na2SO4 and
evaporated to yield 0.618 g (86%) of 16. 1H NMR (500 MHz,
CD3CN): δppm 7.83 (m, 4H, Pht), 7.49-7.46 (m, 2H, DMTr),
7.37-7.30 (m, 6H, DMTr), 7.25-7.21 (m, 1H, DMTr),
6.91-6.86 (m, 4H, DMTr), 4.42 (m, 1H, CHHON), 4.38-4.32
(m, 3H, CHHON, H-1, and H-3), 4.01 (m, 1H, H-4),
3.84-3.67 (m, 8H, POCH2 and 2 × OCH3), 3.60 (m, 2H,
H-2), 3.15 (m, 1H, H-5a), 3.05 (m, 1H, H-5b), 2.66 (t, 1H,
J ) 6.0 Hz, CHHCN), 2.55 (t, 1H, J ) 6.0 Hz, CHHCN),
2.24-2.00 [m, 3H, CH(CH3)3 and CH2CH2ON], 1.93-1.85
[m, 1H, CH(CH3)3], 1.19-1.03 (m, 12H, 4 × CH3); 31P NMR
(200 MHz, CD3CN): δppm 147.2; HRMS (ESI): [M + Na]+
C45H52N3NaO9P requires 832.3339, found 832.3311.
1
CH2Cl2) to yield 1.61 g (82%) of 11 as white powder. H
NMR [500 MHz, (CD3)2SO]: δppm 7.86 (s, 8H, Pht), 7.22
(br s, 1H, Ar), 7.15 (br d, 2H, Ar), 5.16 (s, 4H, 2 × CH2ON),
4.58 (t, 1H, J ) 5.2 Hz, OH), 4.08 (t, 2H, J ) 6.4 Hz,
CH2OAr), 3.56 (dt, 2H, J ) 5.2 and 6.2 Hz, CH2OH), 1.87
(tt, 2H, J ) 6.2 and 6.4 Hz, CH2CH2CH2); 13C NMR [125
MHz, (CD3)2SO]: δppm 163.6, 159.1, 136.4, 135.3, 129.0,
123.7, 122.8, 116.4, 79.3, 65.3, 57.7, 32.5; HRMS (ESI):
[M + Na]+ C27H22N2NaO8 requires 525.1274, found 525.1305.
3-[3,5-Bis(phthalimidoxymethyl)phenoxy]propyl 2-cyano-
ethyl N,N-Diisopropylphosphoramidite (12). Compound 11
(290 mg, 0.578 mmol) was dried over P2O5 and suspended
in dry 1,4-dioxane (3 mL). Dry triethylamine (287 µL, 2.07
mmol) and 2-cyanoethyl N,N-diisopropylphosphonamidic
chloride (141 µL, 0.632 mmol) were added under nitrogen.
The mixture was stirred for 2.5 h and then subjected directly
to a silica gel column. Elution with 1% triethylamine and
29% petroleum ether in ethyl acetate yielded 284 mg (70%)
1
of 12 as white powder. H NMR (500 MHz, CDCl3): δppm
7.81 (m, 4H, Pht), 7.73 (m, 4H, Pht), 7.21 (m, 1H, Ar), 7.16
(br d, 2H, Ar), 5.17 (s, 4H, 2 × CH2ON), 4.15 (t, 2H, J )
6.1 Hz, CH2OAr), 3.91-3.76 (m, 4H, 2 × POCH2), 3.61 [m,
2H, 2 × CH(CH3)2], 2.64 (t, 2H, J ) 6.5 Hz, CH2CN), 2.10
(m, 2H, CH2CH2CH2), 1.19 (s, 3H, CH3), 1.18 (s, 3H, CH3),
1.18 (s, 3H, CH3), 1.17 (s, 3H, CH3); 13C NMR (125 MHz,
CDCl3): δppm 163.4, 159.2, 135.4, 134.4, 128.9, 123.5, 122.7,
117.7, 116.5, 79.5, 64.7, 60.1 [d, J(13C, 31P) ) 17.6 Hz],
58.4 [d, J(13C, 31P) ) 19.2 Hz], 43.0 [d, J(13C, 31P) ) 12.5
Hz], 30.9 [d, J(13C, 31P) ) 7.2 Hz], 24.6 (m), 20.4 [d, J(13C,
31P) ) 7.1 Hz]; 31P NMR (200 MHz, CDCl3): δppm 147.7;
HRMS (ESI): [M + Na]+ C36H39N4NaO9P requires 725.2353,
found 725.2371.
5-(4,4′-Dimethoxytrityl)-3-O-tert-butyldimethylsilyloxy-1,2-
dideoxy-1-C-(2-phthalimidoxyethyl)-ꢀ-D-erythro-pentofura-
nose(14).5-(4,4′-Dimethoxytrityl)-3-O-tert-butyldimethylsilyloxy-
1,2-dideoxy-1-C-(2-hydroxyethyl)-ꢀ-D-erythro-pentofuranose
(40) (13, 1.64 g, 2.84 mmol), N-hydroxyphthalimide (0.557 g,
3.42 mmol), and triphenylphosphine (0.896 g, 3.41 mmol) were
dissolved in dry THF (40 mL). Diethylazodicarboxylate (DEAD,
573 µL, 3.68 mmol) was added dropwise and the mixture was
stirred for 89 h at room temperature. After evaporation, the
residue was dissolved in CH2Cl2 and washed three times with
saturated NaHCO3. The combined aqueous layers were extracted
with CH2Cl2, and then the organic layers were combined, dried
with Na2SO4, and evaporated. Crude product was purified by
Synthesis of Oligodeoxyribonucleotide 5′-Glycoconjugate
(20). The oligodeoxyribonucleotide 18 was synthesized on
an Applied Biosystems 392 DNA synthesizer on 1.0 µmol
scale using commercial 1000 Å CPG-succinyl-thymidine
support and conventional phosphoramidite chemistry. Stan-
dard DNA coupling protocol was used except for a prolonged
coupling time for phosphoramidite 12 (600 s, 0.13 mol L-1
solution in dry MeCN). After the oligonucleotide assembly,
the support-bound material was removed from the synthesizer