S.P. Vincent, L.N. Gastinel / Carbohydrate Research 337 (2002) 1039–1042
1041
distilled over CaH2 and CH2Cl2 over P2O5. NMR spec-
eluant was water with a flow-rate of 1 mL min−1
.
tra were recorded on Bruker AC-250 or AMX-400
apparatus.
Samples containing guanosine derivatives were detected
by a UV-lamp (254 nm). Under these conditions the
final GDP-hexanolamine came out first from the
column, followed by GMP. Fractions containing the
title compound were collected and freeze-fried yielding
141 mg (2.1×10−4 mol, 88% yield) of pure GDP-hex-
anolamine as a white viscous solid.
GMP-salt preparation.—GMP disodium salt was dis-
solved in distilled water (c=0.5 mol l−1) and mixed
with 4 wt equiv of Dowex WX8-200, H+ form, and
shaked for 1 h. After filtration, the resin was abun-
dantly washed with distilled water. 2 equiv of triethy-
lamine in EtOH were added to the GMP aqueous
solution and concentrated to dryness under diminished
pressure. The residual crystalline white powder was
dried further under high vacuum and used as such for
the coupling experiments.
NMR: 1H (400 MHz, D2O): l 8.29 (s, 1 H, H-8), 5.77
(d, J 6.0 Hz, 1 H, H-1%), 4.36 (dd, J 3.5, J 5.1 Hz, 1 H,
H-3%), 4.18 (m, 1 H, H-4%), 4.04 (m, 2 H, H-5%), 3.69 (q,
J 6.7 Hz, 2 H, CH2ꢀOP), 2.77 (t, J 7.3 Hz, 1 H,
CH2ꢀNH2), 1.6–0.9 (m, 8 H); 31P (101 MHz, D2O): l
−12.88 (d, J 21.1 Hz), −13.55 (d, J 21.1 Hz); MS (ES,
negative mode): m/z 541.04 [M−1].
GDP-hexanolamine, disodium salt (1)6
GMP acti6ation. GMP·2 Et3NH+ (120 mg, 2.38
mmol) was suspended under an argon atmosphere into
3 mL of freshly distilled MeCN. At 0 °C, 120 mL of
N,N-dimethylaniline (9.4×10−4 mol, 4.0 equiv) and 50
mL of triethylamine (4.5×10−4 mol, 1.9 equiv) were
added. A precooled solution of 200 mL of trifl-
uoroacetic acid anhydride (1.4×10−3 mol, 5.9 equiv)
in 1 mL anhyd MeCN were added dropwise over 5 min.
The solution turned red–brown and rapidly homoge-
nized. The reaction mixture was stirred for 15 min at
0 °C, concentrated under diminished pressure, and
dried for 10 min under diminished pressure (oil pump).
The polyacylated GMP was then dissolved into 3 mL
anhyd MeCN. At 0 °C, 100 mL of 1-methylimidazole
(1.25 mmol, 5.3 equiv), as well as 130 mL of triethy-
lamine (1.2 mmol, 5.0 equiv) were added. A bright
yellow color then appeared.
1-O-(Dibenzyl phosphoryloxy)-6-trifluoroacetamido-
1-hexanol (2).—6-Trifluoroacetamido-1-hexanol14 (1.0
g, 8.6 mmol, prepared by following
a known
procedure15) was dissolved in 10 mL of anhyd CH2Cl2
(solution A). In a separate flask was added, dropwise at
0 °C, 2.3 mL of oxalyl chloride (26.1 mmol, 3.0 equiv)
into a solution of 3.6 g of dibenzylphosphate (12.9
mmol, 1.5 equiv) and 10 mL anhyd DMF in 60 mL
anhyd CH2Cl2. The reaction mixture was then stirred
for 1 h under Ar. The crude mixture was concentrated
under diminished pressure, diluted in 60 mL anhyd
toluene and concentrated again. The resulting oil was
dissolved in 40 mL anhyd CH2Cl2 and slowly added at
0 °C to solution A (6-trifluoroacetamido-1-hexanol in
10 mL anhyd CH2Cl2) containing 1.2 mL of 1-
methylimidazole (15.0 mmol, 1.7 equiv). The solution
was stirred 1 h at 0 °C, then overnight at rt. The final
solution was washed once with HCl 1 N (25 mL) and
once with 25 mL satd NaHCO3, dried over MgSO4,
and concentrated to dryness under diminished pressure.
The residue was chromatographed on silica gel (gradual
elution 2:1 to 3:2 cyclohexane–EtOAc) to yield 3.3 g
(81% over two steps) of the pure title compound (white
solid).
Coupling to the phosphate 3. To the preceding solu-
,
tion were added 200 mg of preactivated 4 A molecular
sieves. A solution of 6-trifluoroacetamido-hexanol 1-
phosphate triethylammonium salt 3 (54 mg, 1.5×10−4
mol, 0.63 equiv) in 1 mL anhyd MeCN was then added
dropwise (over 1 min) into the reaction mixture. The
mixture was stirred for 1 h at 0 °C under argon, then
for 2 h at rt. The reaction could be monitored by TLC
(10:11:4 CHCl3–MeOH–AcO−,NH4+ (1 M, pH 7)) and
by 31P NMR. After filtration through Celite, the reac-
tion was quenched by 15 mL distilled water. The final
mixture was extracted three times with 15 mL CH2Cl2.
The combined organic phases were washed once by 10
mL of water. The aqueous phases were combined and
concentrated under diminished pressure.
1
NMR H (250 MHz, CDCl3): l 7.50–7.35 (m, 10 H,
Ar), 5.12 (ABX system, JAB 13.4, JAP=JBP 11.8 Hz, 4
H, CH2f), 4.07 (q, J 7.3 Hz, 2 H, CH2ꢀOP), 3.40 (q, J
6.5 Hz, 2 H, CH2ꢀNH2), 1.65 (hept., J 6.7 Hz, 4 H),
1.40 (m, 4 H); 13C (50 MHz, CDCl3): l 157.4 (q, J 36.4
Hz, CꢁO), 135.80, 135.67, 129.12, 128.52, 128.35,
128.10, 127.67, 127.45, 116.05 (q, J 286 Hz, CF3), 69.30
(d, J 5.6 Hz, CH2f), 67.63 (d, J 5.6 Hz, CH2f), 62.43,
39.58, 29.90, 28.57, 25.83, 24.79; 31P (101 MHz, D2O):
l −0.70 (s); MS (DCI-NH3): m/z 491 [M+18].
6-Trifluoroacetamido-hexanol 1-phosphate triethylam-
monium salt (3)6,16.—Phosphate 2 (210 mg 4.44×10−4
mol) was dissolved in 4 mL MeOH and 3 mL MeOAc.
Triethylamine (60 mL, 1.0 equiv) and 49 mg Pd/C (10%)
were added and the resulting suspension was vigorously
stirred overnight under H2 (0.1 HPa). The final mixture
was filtrated through a short pad of Celite. The filtrate
Trifluoroacetamide deprotection. The crude reaction
mixture was dissolved in 20 mL NH4OH (3 M) and
stirred for 4 h at rt. After concentration by rotary
evaporation, the crude mixture was dissolved in 5 mL
of water and shaked with 1 g of Dowex 200WX8 (Na+
form) for 1 h.
Purification. The crude mixture was dissolved in 2
mL of water and passed through a Sephadex G25
8
column (2.5×26 cm) using an FPLC apparatus (Akta,
Pharmacia Biotech). Chromatography conditions: the