Mechanistic InVestigation of UDP-Galactopyranose Mutase
J. Am. Chem. Soc., Vol. 123, No. 28, 2001 6759
resolution FABMS calcd for C6H12FO8PNa [M + Na]+ 285.0152, found
m/z 285.0152.
was kept at 75 °C for 6 h. At the end of the incubation, solvent was
evaporated under reduced pressure. The residue was chromatographed
on a silica gel column which was eluted with EtOAc/hexanes (1:1).
The desired product was isolated as a mixture of R and â anomers in
71% total yield (3.1 g). Spectral data for this mixture: 1H NMR (CDCl3)
δ 2.05 (3H, s), 2.06 (3H, s), 2.10 (3H, s), 2.11 (3H, s), 4.08-4.20
(3H, m), 4.25-4.35 (4H, m), 4.48 (1H, ddd, J ) 24.0, 4.5, 3.5, 4-H),
4.79 (1H, ddd, J ) 52.5, 3.5, 1.5, 3-H), 4.95 (1H, dt, J ) 54, 4.5,
3-H), 5.26-5.37 (4H, m); 13C NMR (CDCl3) δ 20.70, 20.72, 20.9,
21.0, 62.4, 62.6, 70.2 (d, J ) 6), 71.6 (d, J ) 7), 75.3 (d, J ) 23), 78.5
(d, J ) 27), 79.3 (d, J ) 24), 80.9 (d, J ) 28), 96.5 (d, J ) 7), 96.8
(d, J ) 184), 97.0 (d, J ) 185), 102.4 (d, J ) 4), 171.0, 171.2, 171.31,
171.33; 19F NMR (CDCl3) δ -194.4 (ddd, J ) 53.6, 23.4, 18.9), -186.2
(ddd, J ) 52.8, 24.0, 15.2); high-resolution CIMS calcd for C10H19-
FNO7 [M + NH4]+ 284.1146, found m/z 284.1169.
1,2,5,6-Tetra-O-acetyl-3-deoxy-3-fluoro-r,â-D-galactofuranose (22).
A solution of 21 (3.1 g, 12 mmol, a mixture of R and â anomers) in
pyridine (10 mL) and acetic anhydride (10 mL) was stirred at room
temperature for 2 h. Removal of solvent to dryness under reduced
pressure gave the desired product 22 as a mixture of R and â anomers
in quantitative yield (3.78 g). This mixture was pure enough to be used
in the next step of synthesis without purification: 1H NMR (CDCl3) δ
2.08 (3H, s), 2.09 (3H, s), 2.10 (3H, s), 2.12 (3H, s), 2.13 (9H, s), 2.15
(3H, s), 4.13 (1H, dd, J ) 12.5, 6.0), 4.19 (1H, dd, J ) 11.5, 7.0),
4.29 (1H, ddd, J ) 20.5, 7.5, 5.5), 4.34 (1H, dd, J ) 12.0, 4.0), 4,35
(1H, dd, J ) 12.0, 3.5), 4.52 (1H, dt, J ) 23.5, 4.0), 4.93 (1H, ddt, J
) 51.3, 4.2, 0.9), 5.21 (1H, ddd, J ) 56.0, 6.5, 5.5), 5.25-5.40 (4H,
m), 6.21 (1H, s), 6.36 (1H, d, J ) 4.5); 13C NMR (CDCl3) δ 20.4,
20.60, 20.66, 20.69, 20.79, 20.81, 21.0 (2C), 62.0, 62.2, 69.1 (d, J )
8), 70.5 (d, J ) 5), 75.6 (d, J ) 23), 78.6 (d, J ) 27), 80.0 (d, J ) 29),
82.6 (d, J ) 28), 92.9 (d, J ) 10), 93.3 (d, J ) 190), 94.8 (d, J )
187), 98.8 (d, J ) 3), 169.0, 169.1, 169.2, 169.6, 170.0, 170.1, 170.5;
19F NMR (CDCl3) δ -199.4 (dt, J ) 55.9, 21.2), -188.6 (ddd, J )
51.9, 24.0, 18.2); high-resolution FABMS calcd for C14H19FO9Na [M
+ Na]+ 373.0911, found m/z 373.0924.
Dibenzyl (2,5,6-Tri-O-acetyl-3-deoxy-3-fluoro-r-D-galactofura-
nosyl)phosphate (23r) and Dibenzyl (2,5,6-Tri-O-acetyl-3-deoxy-
3-fluoro-â-D-galactofuranosyl)phosphate (23â). A solution of 22 (2.0
g, 5.7 mmol) in anhydrous dichloromethane (10 mL) was cooled with
an ice-water bath. To this solution was slowly added bromotrimeth-
ylsilane (5 mL), and the mixture was stirred at room temperature for
18 h. The solution was evaporated to dryness under reduced pressure.
Anhydrous toluene (5 mL) was then added and dried in vacuo to
facilitate the evaporation of more volatile silane derivatives. The
resulting dry residue and dibenzyl phosphate (2.4 g, 8.4 mmol) was
redissolved in anhydrous toluene (10 mL). To this solution was added
triethylamine (1.2 mL, 8.4 mmol), and the mixture was stirred over-
night at room temperature. After removal of solvent in vacuo, the
residue was chromatographed on a silica gel column which was eluted
with EtOAc/hexanes (1:1). The fast-moving unreacted starting material
(724 mg, 36%) was recovered. The R isomer of the desired product
was eluted next from the column and was further purified on silica gel
with CHCl3/methanol (99:1) to give pure 23r in 8% yield (252 mg).
The next-eluted component was the â isomer of the desired product
(23â) that was isolated in 17% yield (539 mg). Spectral data for the R
isomer 23r: 1H NMR (CDCl3, 500 MHz) δ 1.96 (3H, s), 2.01 (3H, s),
2.05 (3H, s), 4.13 (1H, dd, J ) 12.0, 6.5, 6-H), 4.32 (1H, dd, J ) 12.0,
4.5, 6-H), 4.34 (1H, dt, J ) 20.0, 7.0, 4-H), 5.04 (4H, d, J ) 7.5,
benzylic CH2’s), 5.15 (1H, dt, J ) 56.5, 7.0, 3-H), 5.26 (1H, dddd, J
) 21.0, 7.0, 4.5, 2.5, 2-H), 5.29 (1H, m, 5-H), 5.99 (1H, dd, J ) 7.0,
4.5, 1-H), 7.34 (10H, m, Ar-H’s); 13C NMR (CDCl3, 125 MHz) δ 20.2,
20.6 (2C), 61.9, 69.4 (2C, d, J ) 6), 69.8 (d, J ) 3), 75.8 (dd, J ) 23,
7), 78.6 (d, J ) 26), 91.9 (d, J ) 188), 97.0 (dd, J ) 10, 5), 127.9
(2C), 128.0 (2C), 128.61 (2C), 128.63 (3C), 128.7, 135.5 (d, J ) 7),
135.6 (d, J ) 7), 169.8, 170.1, 170.4; 31P NMR (CDCl3) δ -2.5; 19F
NMR (CDCl3) δ -203.7 (dt, J ) 56.4, 20.3); high-resolution FABMS
calcd for C26H31FO11P [M + H]+ 569.1588, found m/z 569.1633.
Spectral data for the â isomer 23â: 1H NMR (CDCl3) δ 2.02 (3H, s),
2.07 (3H, s), 2.11 (3H, s), 4.10 (1H, dd, J ) 12.0, 6.9, 6-H), 4.24 (1H,
dd, J ) 12.0, 4.0, 6-H), 4.49 (1H, dt, J ) 24.2, 4.0, 4-H), 4.89 (1H,
dd, J ) 51.6, 4.0, 3-H), 5.07 (2H, d, J ) 8.0, benzylic CH2), 5.08 (2H,
Bis(triethylammonium) UDP-[2-F]Galf (9). Compound 16 (310
mg, 0.67 mmol) was dried by repetitive coevaporation with anhydrous
pyridine (3 mL). To this residue was added UMP-morpholidate (1.2 g,
1.7 mmol) in anhydrous pyridine (3 mL), and the solution was
evaporated again to dryness under vacuum. This was followed by the
addition of 1H-tetrazole (155 mg, 2.0 mmol) in anhydrous pyridine (3
mL), and the resulting solution was stirred at room temperature for 34
h. Removal of the solvent under vacuum gave a solid residue which
was dissolved in methanol and chromatographed on a Sephadex LH-
20 column (2.5 × 120 cm) using methanol as the eluent. Fractions
containing the desired product, judging by TLC analysis (EtOH:
NH4OH:H2O ) 5:3:1), were pooled and evaporated to dryness under
reduced pressure. Compound 9 was further purified by HPLC using a
C18 column (10 × 250 mm). The eluent was 1% aqueous acetonitrile
in 50 mM triethylammonium acetate, pH 6.8, and the flow rate was 5
mL/min. The retention time of 9 under these conditions was 14.4 min.
The yield was 43% (70 mg) after lyophilization: 1H NMR (D2O, 500
MHz) δ 1.30 (18H, t, J ) 7.2, Et3N-Me), 3.22 (12H, q, J ) 7.2, Et3N-
CH2), 3.66 (1H, dd, J ) 12.0, 7.0, 6-H), 3.73 (1H, dd, J ) 12.0, 5.0,
6-H), 3.82 (1H, dt, J ) 7.0, 5.0, 5-H), 3.89 (1H, dd, J ) 7.0, 5.0,
4-H), 4.20-4.26 (2H, m, 5′-H2), 4.28-4.31 (1H, m, 4′-H), 4.36-4.41
(2H, m, 2′-H, 3′-H), 4.57 (1H, dt, J ) 17.5, 7.0, 3-H), 5.05 (1H, dddd,
J ) 53.0, 7.0, 5.3, 1.5, 2-H), 5.79 (1H, t, 5.3, 1-H), 5.97 (1H, d, J )
8.0, 5′′-H), 6.03 (1H, d, J ) 4.5, 1′-H), 7.94 (1H, d, J ) 8.0, 6′′-H);
13C NMR (D2O) δ 9.8 (Et3N-Me), 48.1 (Et3N-CH2), 63.5 (C-6), 66.5
(d, J ) 5, C-5′), 71.2 (C-2′), 73.2 (d, J ) 21, C-3), 73.3 (C-5), 75.2
(C-3′), 82.6 (d, J ) 10, C-4), 84.9 (d, J ) 9, C-4′), 89.8 (C-1′), 95.7
(dd, J ) 198, 7, C-2), 96.8 (dd, J ) 18, 5, C-1), 104.2 (C-5′′), 143.3
(C-6′′), 153.3 (C-2′′), 167.6 (C-4′′); 31P NMR (D2O) δ -11.1 (d, J )
20), -12.8 (d, J ) 20); 19F NMR (D2O) δ -204.6 (dt, J ) 52.8, 18.1);
high-resolution FABMS calcd for C15H23FN2O16P2Na [M + Na]+
591.0405, found m/z 591.0441.
Synthesis of UDP-[3-F]Galf (10). 1,2-O-Isopropylidene-3-deoxy-
3-fluoro-r-D-galactofuranose (19). 1,2:5,6-Di-O-isopropylidene-3-
deoxy-3-fluoro-R-D-galactofuranose (18, 4.7 g, 18 mmol), which was
prepared from 17 by a literature procedure,13 was dissolved in 80%
aqueous acetic acid (40 mL) and kept at 70 °C for 25 min. The mixture
was evaporated to dryness under reduced pressure, and the residual oil
was subjected to chromatography on silica gel and eluted with EtOAc/
hexanes (1:1). The desired product 19 was isolated in 93% yield (3.7
g): 1H NMR (CDCl3) δ 1.33 (3H, s), 1.52 (3H, s), 2.24 (1H, s, exch.),
2.89 (1H, s, exch.), 3.66 (1H, dd, J ) 11.7, 4.5, 6-H), 3.78 (1H, dd, J
) 11.7, 3.9, 6-H), 3.86 (1H, dddd, J ) 7.5, 4.5, 3.9, 0.9, 5-H), 4.31
(1H, dddd, J ) 24.0, 7.5, 1.8, 0.9, 4-H), 4.78 (1H, dd, J ) 15.3, 4.2,
2-H), 5.00 (1H, dd, J ) 51.0, 1.8, 3-H), 5.98 (1H, d, J ) 4.2, 1-H);
13C NMR (CDCl3) δ 25.9, 26.5, 63.4, 69.9 (d, J ) 9), 84.4 (d, J )
32), 85.9 (d, J ) 25), 95.3 (d, J ) 182), 105.6, 113.5; 19F NMR (CDCl3)
δ -187.3 (ddd, J ) 50.1, 24.1, 15.0); high-resolution CIMS calcd for
C9H19FNO5 [M + NH4]+ 240.1247, found m/z 240.1263.
5,6-Di-O-acetyl-1,2-O-isopropylidene-3-deoxy-3-fluoro-r-D-ga-
lactofuranose (20). A solution of 19 (3.7 g, 17 mmol) in pyridine (10
mL) and acetic anhydride (10 mL) was stirred at room temperature for
2 h. Removal of the solvent under reduced pressure afforded the
acetylated product 20 in quantitative yield (5.1 g). The product was
pure enough to be used in the next step without purification: 1H NMR
(CDCl3) δ 1.34 (3H, s), 1.57 (3H, s), 2.06 (3H, s), 2.10 (3H, s), 4.17
(1H, dd, J ) 12.2, 5.8, 6-H), 4.30 (1H, ddd, J ) 24.8, 7.0, 3.0, 4-H),
4.36 (1H, dd, J ) 12.2, 4.0, 6-H), 4.73 (1H, dd, J ) 14.2, 3.8, 2-H),
4.96 (1H, dd, J ) 50.8, 3.0, 3-H), 5.34 (1H, ddd, J ) 7.0, 5.8, 4.0,
5-H), 5.90 (1H, d, J ) 3.8, 1-H); 13C NMR (CDCl3) δ 20.7, 20.9, 26.3,
26.7, 62.7, 69.7 (d, J ) 9), 82.6 (d, J ) 28), 84.2 (d, J ) 32), 94.1 (d,
J ) 182), 105.1, 114.0, 170.1, 170.5; 19F NMR (CDCl3) δ -187.7
(ddd, J ) 50.8, 25.0, 14.5); high-resolution FABMS calcd for C13H20-
FO7 [M + H]+ 307.1193, found m/z 307.1181.
5,6-Di-O-acetyl-3-deoxy-3-fluoro-r,â-D-galactofuranose (21). A
solution of 20 (5.0 g, 16 mmol) in 80% aqueous acetic acid (100 mL)
(13) (a) Brimacombe, J. S.; Foster, A. B.; Hem, R.; Westwood, J. H.;
Hall, L. D. Can. J. Chem. 1970, 48, 3946-3952. (b) Kovac, P.; Glaudemans,
C. P. J. Carbohydr. Res. 1983, 123, 326-331.