Notes
J . Org. Chem., Vol. 67, No. 12, 2002 4375
m), 7.93 (8 H, m), 8.60 (1 H, s), 8.69 (1 H, s), 8.74 (1 H, s), 8.75
(1 H, s), 9.41 (1 H, s), 9.49 (1 H, s); 13C NMR (CDCl3, 75.4 MHz)
being concentrated to dryness under reduced pressure. The
resulting white solid was mixed with water (5 mL) and filtered
through paper to remove insoluble material. Concentration of
the filtrate gave a crude product that was purified on Sephadex
G-10 (eluant: H2O) to afford the ammonium salt of the target
N-acylphosphoramidate 2. Further purification by reverse phase
HPLC with gradient elution (C18 column: solvent flow rate 2
mL/min; from 100:0 H2O: MeOH + 0.1% TFA to 85:15 H2O:
MeOH + 0.1% TFA over a period of 65 min), with monitoring
260 nm, gave the trifluoroacetate salt of 2 (ret time: 104 min)
2
2
δ 62.10 (dt, J CP ) 10.5 Hz), 62.22 (dt, J CP ) 10.5 Hz), 64.86
2
(dt, J CP ) 10.5 Hz), 71.76 (d), 71.93 (d), 74.18 (d), 74.49 (d),
3
3
82.53 (dt, J CP ) 4.7 Hz), 82.71 (dt, J CP ) 4.7 Hz), 85.45 (d),
85.59 (d), 123.08 (s), 127.46 (d), 127.89 (d), 127.92 (d), 128.09
(d), 128.23 (d), 128.37 (d), 128.43 (d), 128.52 (d), 129.62 (d),
132.48 (s), 133.49 (s), 137.20 (s), 137.22 (s), 141.33 (d), 141.62
(d), 149.51 (s), 151.81 (d), 151.90 (d), 152.66 (s), 164.81 (s), 164.91
(s), 165.20 (s); 31P NMR (CDCl3, 121 MHz) δ 141.17 (s); MS (FAB)
1317 (MNa+, 2), 1295 (MH+, 1), 281 (10), 240 (38), 201 (100);
exact mass calcd for MNa+ C69H55N10O15PNa requires 1317.3484,
found 1317.3464 (FAB).
1
as a white solid after freeze-drying overnight: 67 mg, 40%; H
NMR (D2O, 300 MHz) δ 2.78 (2 H, m), 3.82 (2 H, m), 3.99 (1 H,
t, J ) 5.1 Hz), 4.04 (1 H, m), 4.11 (1 H, m), 4.40 (1 H, m), 5.83
(1 H, m), 8.10 (1 H, s), 8.30 (1 H, s); 13C NMR (D2O, 75.4 MHz)
Samples of the starting materials 8 (210 mg) and 5 (190 mg)
were also recovered from the column.
2
δ 48.59 (t), 64.20 (dt, J CP ) 4.8 Hz), 69.80 (d), 74.14 (d), 83.38
1
(d), 83.50 (d), 87.45 (d), 115.72 (q, J CF ) 290.0 Hz), 117.95 (s),
O-(6-N-Ben zoyl-2′,3′-O-d ib en zoyla d en osin e-5′-O-yl)-N-
(O-b en zyl-N-b en zyloxyca r b on yl-L-a sp a r a gin yl)-O-b en z-
yloxyp h osp h or a m id a t e (12). The N-acylphosphoramidite 9
(369 mg, 0.34 mmol) was dissolved in anhydrous CH2Cl2 (5 mL)
and degassed with Ar before being cooled to 0 °C. A solution of
t-BuOOH (0.34 mL of a 5 M solution in anhydrous CH2Cl2, 5
equiv) was added slowly at 0 °C, and the reaction mixture was
stirred for 15 min, after which time TLC analysis showed
complete consumption of starting material. The solution was
then concentrated under reduced pressure, and the residue was
purified by chromatography (eluant: 50:1 CHCl3/i-PrOH) to
afford N-acylphosphoramidate 12 as a white foam: 330 mg, 88%;
1H NMR (CDCl3, 300 MHz) δ 3.00 (2 H, m), 4.51-5.17 (10 H,
m), 6.00 (1 H, m), 6.10 (1 H, m), 6.55 (1 H, d, J ) 8.4 Hz), 6.61
(1 H, d, J ) 6.2 Hz), 6.66 (1 H, d, J ) 6.0 Hz), 7.15-7.55 (24 H,
m), 7.85 (2 H, m), 7.97 (4 H, m), 8.74 (1 H, s), 8.79 (1 H, s), 9.73
(1 H, br. s); 13C NMR (CDCl3, 75.4 MHz) δ 38.49 (t), 50.41 (d),
2
141.97 (d), 144.11 (s), 147.81 (d), 149.31 (s), 162.30 (q, J CF
)
35.4 Hz), 170.46 (s), 172.08 (s); 31P NMR (D2O, 121 MHz) δ
-5.17; 19F NMR (D2O, 282.2 MHz) δ -76.22; MS (FAB) 462
(MH+, 3), 217 (12), 149 (14), 138 (27), 136 (74), 133 (100); exact
mass calcd for MH+ C14H21N7O9P requires 462.1138, found
462.1139 (FAB).
O-(6-N-Ben zoyl-2′,3′-O-d ib en zoyla d en osin e-5′-O-yl)-N-
(O-b en zyl-N-b en zyloxyca r b on yl-D-a sp a r a gin yl)-O-b en z-
yloxyp h osp h or a m id a te (17). The protected derivative of D-
asparagine 14 (782 mg, 2.2 mmol) was coupled to the protected
adenosine 8 (1.65 g, 2.8 mmol) and the activated phosphine 6
(0.36 mL, 2.2 mmol), using our general “one-pot” procedure, and
oxidation of the resulting N-acylphosphoramidite intermediate
16 was accomplished by treatment with t-BuOOH to give the
protected N-acylphosphoramidate 17 as a white foam: 711 mg,
30% (based on 14); 1H NMR (CDCl3, 300 MHz) δ 3.00 (2 H, m),
4.57 (2 H, m), 4.80-5.15 (6 H, m), 6.00 (2 H, m), 6.13 (1 H, m),
6.26 (1 H, m), 6.60 (1 H, d, J ) 6.6 Hz), 6.68 (1 H, d, J ) 6.6
Hz), 6.70 (1 H, d, J ) 7.2 Hz), 7.10-7.54 (24 H, m), 7.80-7.99
(6 H, m), 8.81 (1 H, s), 8.82 (1 H, s), 9.78 (1 H, br s); 13C NMR
2
2
66.63 (dt, J CP ) 13.0 Hz), 66.64 (t), 67.11 (t), 69.89 (dt, J CP
)
2
5.6 Hz), 71.49 (d), 74.18 (d), 81.81 (d, J CP ) 7.0 Hz), 85.20 (d),
122.76 (s), 127.62 (d), 127.72 (d), 127.76 (d), 127.85 (d), 127.97
(d), 128.05(d), 128.08 (d), 128.19 (d), 128.22 (d), 128.29 (d), 128.33
(d), 128.38 (d), 129.63 (d), 129.65 (d), 132.40 (s), 133.58 (s), 134.74
2
(CDCl3, 75.4 MHz) δ 38.89 (t), 50.29 (d), 66.48 (dt, J CP ) 10.7
Hz), 66.96 (t), 69.52 (t), 71.43 (dt, 2J CP ) 8.9 Hz), 73.83 (d), 74.03
(d), 81.79 (d), 84.84 (d), 122.68 (s), 127.38 (d), 127.52 (d), 127.71
(d), 127.89 (d), 127.93 (d), 128.02 (d), 128.08 (d), 128.12 (d),
128.26 (d), 128.39 (d), 129.51 (d), 132.21 (s), 132.32 (s), 133.47
(s), 134.74 (s), 135.07 (s), 135.89 (s), 141.65 (d), 149.61 (s), 151.65
(d), 152.61 (s), 156.16 (s), 164.63 (s), 164.71 (s), 164.99 (s), 170.87
(s), 172.42 (s); 31P NMR (CDCl3, 121 MHz) δ -1.06, -2.18; MS
(FAB) 1088 (MH+, 8), 240 (14), 201 (98), 105 (26), 91 (100); exact
mass calcd for MH+ C57H51N7PO14 requires 1088.3232, found
1088.3220 (FAB).
2
(s), 134.83 (s), 135.06 (d, J CP ) 6.0 Hz), 135.91 (s), 141.76 (d),
149.63 (s), 151.71 (d), 152.67 (s), 156.02 (s), 164.80 (s), 165.11
(s), 170.88 (s), 170.95 (s), 172.44 (d, J CP ) 4.5 Hz); 31P NMR
2
(CDCl3, 121 MHz) δ -1.44, -1.69 (s); MS (FAB) 1110 (MNa+,
4), 240 (22), 201 (61), 105 (80), 91 (100); exact mass calcd for
MNa+ C57H50N7O14PNa requires 1110.3051, found 1110.3077
(FAB).
Continued elution gave a sample of the symmetrically coupled
product 13 (formed by oxidation of a small amount of 10 in the
starting material) as a white foam; 1H NMR (CDCl3, 300 MHz)
3
O-(Ad en osin e-5′-O-yl)-N-(D-a sp a r a gin yl)p h osp h or a m i-
d a te Tr iflu or oa ceta te (18). The protected derivative 17 (230
mg, 0.2 mmol) was converted to N-acylphosphoramidate 18 using
an identical procedure to that described for the preparation of
2 from 12. The trifluoroacetate salt of the desired compound was
obtained as a white solid after C18 RP-HPLC purification: 51
δ 4.52 (4 H, m), 4.69 (2 H, m), 5.18 (2 H, d, J HP ) 9.6 Hz), 6.08
(2 H, m), 6.19 (2 H, m), 6.62 (2 H, d, J ) 6.0 Hz), 7.24-7.55 (23
H, m), 7.72-7.99 (12 H, m), 8.52 (1 H, s), 8.54 (1 H, s), 8.75 (1
H, s), 8.77 (1 H, s), 9.31 (1 H, br. s), 9.33 (1 H, br. s); 13C NMR
2
(CDCl3, 75.4 MHz) δ 66.58 (br. dt), 70.15 (dt, J CP ) 5.7 Hz),
3
71.02 (d), 71.06 (d), 73.83 (d), 74.11 (d), 81.28 (dt, J CP ) 4.8
1
3
mg, 45%; H NMR (D2O, 300 MHz) δ 2.78 (2 H, m), 3.81 (2 H,
Hz), 81.38 (dt, J CP ) 4.8 Hz), 85.86 (d), 123.19 (s), 123.26 (s),
m), 4.01 (2 H, m), 4.11 (1 H, t, J ) 4.5 Hz), 4.36 (1 H, dd, J )
5.1, 4.8 Hz), 5.82 (1 H, d, J ) 4.8 Hz), 8.08 (1 H, s), 8.29 (1 H,
127.79 (d), 127.94 (d), 127.97 (d), 127.99 (d), 128.16 (d), 128.25
(d), 128.29 (d), 128.32 (d), 128.37 (d), 128.40 (d), 128.47 (d),
128.62 (d), 129.52 (d), 132.35 (s), 133.36 (s), 133.45 (s), 134.91
(s), 134.99 (s), 141.54 (d), 149.55 (s), 151.62 (d), 152.50 (s), 152.58
(s), 164.69 (s), 164.76 (s), 164.82 (s), 164.87 (s), 165.01 (s), 165.04
(s); 31P NMR (CDCl3, 121 MHz) δ -0.50; MS (FAB) 1333 (MNa+,
2
s); 13C NMR (D2O, 75.4 MHz) δ 48.54 (t), 64.14 (dt, J CP ) 5.0
Hz), 69.67 (d), 74.25 (d), 83.26 (d), 83.39 (d), 87.59 (d), 115.64
1
(q, J CF ) 290.0 Hz), 117.92 (s), 142.00 (d), 144.06 (s), 147.71
2
(d), 149.28 (s), 162.17 (q, J CF ) 35.6 Hz), 170.28 (s), 172.08 (s);
31P NMR (D2O, 121 MHz) δ -5.13; 19F NMR (D2O, 282.2 MHz)
δ -76.23; MS (FAB) 462 (MH+, 6), 263 (14), 217 (72), 136 (42),
133 (13); exact mass calcd for MH+ C14H21N7PO9 requires
462.1138, found 462.1150 (FAB).
3), 240 (28), 201 (100); exact mass calcd for MNa+ C69H55N10
PO16Na requires 1333.3433, found 1333.3477 (FAB).
-
O-(Ad en osin e-5′-O-yl)-N-(L-a sp a r a gin yl)p h osp h or a m i-
d a te Tr iflu or oa ceta te (2). The fully protected N-acylphospho-
ramidate 12 (345 mg, 0.32 mmol) was suspended in a solution
of MeOH (6 mL) and EtOH (2 mL) containing 1,4-cyclohexadiene
(0.91 mL, 9.6 mmol). After the addition of 10% Pd/C (960 mg),
the reaction mixture was vigorously stirred at 30 °C for 4 h,
before the addition of further portions of 10% Pd/C (400 mg) and
1,4-cyclohexadiene (0.60 mL). Stirring was then continued for
another 3 h at this temperature. Distilled water (5 mL) was then
added, and the suspension was filtered through Celite. The solid
residues were washed well using 50% aq MeOH, and the filtrate
was combined with the washings. Removal of the solvent then
gave a white solid that was dissolved in concentrated NH4OH
(20 mL) and freshly distilled 1,4-dioxane (20 mL). This solution
was stirred for 6 h at RT, under an inert N2 atmosphere, before
Ack n ow led gm en t. We thank the National Insti-
tutes of Health (NIH), National Cancer Institute
(CA28725), the Florida Department of Health (BMP-
006), and the University of Florida Biotechnology Pro-
gram for funding.
Su p p or tin g In for m a tion Ava ila ble: 1H, 13C, and 31P
NMR spectra of 2, 12, 13, 17, and 18, 19F spectra of 2 and 18,
together with 1H and 13C spectra for 8 and 11. This material
J O025510L