1836
F. Schweizer / Carbohydrate Research 342 (2007) 1831–1840
tography using 3% MeOH in CH2Cl2 as eluent to afford
11 (45 mg, 80%) which was directly used for the cou-
pling to 6.
P2 column (Bio Rad) to afford compound 8 (800 mg,
1.16 mmol, 51%). 1H NMR (D2O): d 0.72 (t, 3H,
CH2CH2CH3), 1.30–1.40 (m, 2H, CH2CH2CH3), 2.50–
2.68 (m, 4H, –CH2CH2CO2NH–), 2.92–3.01 (m, 3H,
2 · ArCH2, –CONHCH2), 3.10–3.16 (m, 1H, –CON-
HCH2), 3.98–4.04 (m, 2H, H05a, H50b), 4.21–4.25 (m,
1H, H-40), 4.31–4.35 (m, 1H, H-30), 4.36–4.41 (m, 2H,
H-20, Ha(Tyr(NO2))), 5.93 (d, 1H, J = 5.5 Hz, H-10),
6.94 (d, 1H, J = 8.6 Hz, Ar-H), 7.36 (d, 1H,
J = 8.6 Hz, Ar-H), 7.74 (s, 1H, Ar-H), 7.82 (s, 1H, Ar-
H); 13C NMR (D2O): d 10.5, 15.3, 15.4, 34.0, 36.0,
41.1, 55.4, 65.4 (d, JC,P = 6.5 Hz), 70.2, 73.6, 84.0 (d,
JC,P = 7.5 Hz), 88.3, 113.6, 122.8, 125.0, 125.9, 135.2,
137.7, 138.2, 151.7, 158.8, 165.5, 172.8, 175.1; ESIMS
m/z calcd for [C24H32N5O14P+Na]+: 668.1581; found,
668.1587.
4.5. 20,30-O-Isopropylidene uridine-5-(ethylcarboxamide
N-(Tyr-(3-nitro)) N0-propylamide) (7)
Compound 6 (1.00 g, 2.81 mmol) and compound 11
(0.98 g, 3.65 mmol) and TBTU (1.17 g, 3.64 mmol) were
dissolved in DMF (20 mL) and diisopropylethylamine
(733 lL) was added. After 2 h the solvent was removed
under reduced pressure and the crude residue was codis-
tilled with toluene (3 · 30 mL). The product was purified
by column chromatography using 3% MeOH in CH2Cl2
as eluent to afford compound 7 (1.38 g, 2.28 mmol,
1
81%). H NMR (DMSO): d 0.78 (t, 3H, CH2CH2CH3),
1.35 (s, 3H, Me), 1.35–1.45 (m, 2H, CH2CH2CH3), 1.55
(s, 3H, Me), 2.52–2.67 (m, 4H, –CH2CH2CO2NH–),
2.88 (dd, 1H, J = 6.1 Hz, J = 13.6 Hz, ArCH2–), 2.94–
3.03 (m, 2H, ArCH2–, –CONHCH2), 3.26–3.29 (m,
1H, CONHCH2), 3.82 (dd, 1H, J = 2.9 Hz,
J = 12.5 Hz, H-50a), 3.92 (dd, 1H, 2.0 Hz, J = 12.5 Hz,
H-50b), 4.20 (s, 1H, OH, exchanges with D2O), 4.30
(m, 1H, H-40), 4.46 (m, 1H, Ha(Tyr(NO2))), 4.85 (dd,
1H, J = 3.1 Hz, J = 6.3 Hz, H-20), 4.93 (1H, dd,
J = 6.3 Hz, 2.6 Hz, H-30), 5.86 (d, 1H, J = 3.1 Hz, H-
10), 6.30 (br t, 1H, NH, exchanges with D2O), 7.02 (d,
1H, J = 8.6 Hz, Ar-H), 7.39 (dd, 1H, J = 2.1 Hz,
J = 8.6 Hz), 7.50 (s, 1H, Ar-H), 7.95 (dd, 1H,
J = 2.1 Hz, Ar-H), 8.09 (br d, 1H, NH, exchanges with
D2O), 10.1 (br s, 1H, exchanges with D2O), 10.5 (br s,
1H exchanges with D2O); ESIMS [M+H]+: 606.2. Anal.
Calcd for C27H35N5O11: C, 53.55; H, 5.83; N, 11.56.
Found: C, 53.21, H, 5.74; N, 11.39.
4.7. Sodium 20,30-O-isopropylidene-50-O-monophospho-
morpholidate uridine-5-(ethylcarboxamide N-Tyr-(3-
nitro) N0-propylamide) (9)
Compound 8 (disodium form, 85 mg, 0.131 mmol) was
passed through a BIO Rad AG 50W-X2 cation ex-
change column (H+, 1.5 · 10 cm) and the solution was
concentrated. The residue was dissolved in a 1:1 mixture
of water (2 mL) and tert-butanol (2 mL), morpholine
(50 lL, 0.58 mmol) and N,N0-dicyclohexylcarbodiimide
(105 mg, 0.51 mmol) was added and the reaction mix-
ture was refluxed for 2 h. TLC (isopropanol/1 M ammo-
nium acetate, 2:1) showed complete conversion into a
higher moving spot. The solution was filtered and parti-
tioned with ether. The aqueous layer was dried under
reduced pressure, dissolved in water and passed through
a BIO Rad AG 50W-X2 cation exchange column (Na+,
1.5 · 10 cm). Evaporation of the solvent under reduced
pressure provided 9 (86 mg, 89%) that was directly used
4.6. Disodium 20,30-O-isopropylidene-50-O-phosphate
uridine-5-(ethylcarboxamide N-(Tyr-(3-nitro)) N0-
propylamide) (8)
1
for the next reaction. H NMR (D2O): d 0.66 (t, 3H,
–CH2CH2CH3), 1.22–1.35 (m, 2H, –CH2CH2CH3),
2.46–2.62 (m, 4H, –CH2CH2CO2NH–), 2.82 (dd, 1H,
J = 8.7 Hz, J = 13.6 Hz, ArCH2–), 2.86–2.95 (m, 2H,
ArCH2–, –CONHCH2–), 3.01–3.06 (m, 4H, 2 ·
–NCH2CH2O–), 3.07–3.13 (m, 1H, –CONHCH2–),
3.60–3.70 (m, 4H, 2 · –NCH2CH2O–), 3.96–4.08 (m,
2H, H50a, H50b), 4.17–4.22 (m, 1H, H-40), 4.28 (dd,
1H, J = 5.3 Hz, J = 5.5 Hz, H-30), 4.31–4.35 (m, 2H,
H-20, Ha(Tyr(NO2))), 5.95 (d, 1H, J = 5.3 Hz, H-10),
6.72 (d, 1H, J = 8.9 Hz, Ar-H), 7.17 (dd, 1H,
J = 2.4 Hz, 8.9 Hz, Ar-H), 7.49 (s, 1H, Ar-H), 7.70 (d,
1H, J = 2.4 Hz, Ar-H); 31P NMR (D2O, H3PO4): d
3.40 (s); ESIMS m/z calcd for [C28H39N6O14P+Na]+:
737.2159; found, 737.2162.
Compound 7 (1.38 g, 2.27 mmol) was dissolved in trieth-
ylphosphate (10 mL) and the reaction mixture was
cooled to 0 ꢁC. Phosphoryl chloride (POCl3, 850 mL,
9 mmol) was slowly added and the ice bath was removed
immediately after addition. After 16 h water (100 mL)
and ether (50 mL) were added. The aqueous layer was
concentrated to dryness and the crude solid residue
was dissolved in 2 M hydrochloric acid (60 mL) and left
for 18 h. Solid sodium bicarbonate was added to neu-
tralize the pH and the solution was evaporated to dry-
ness. The crude material was desalted on a C-18
column using H2O ! 50% MeOH/H2O as eluent. The
combined fractions containing a yellow material were
concentrated. Subsequently, the material was converted
into the disodium salt using the AG 50W-X2 resin (Bio
Rad). The collected yellow fractions were filtered
through a paper filter concentrated and purified by a
4.8. Synthesis of UDP-Gal analogue (2)
Dipotassium-a-D-galactosyl phosphate analogue 18
(42 mg, 125 lmol) was passed through a BIO Rad AG