10498 J. Am. Chem. Soc., Vol. 122, No. 43, 2000
Hallis et al.
(C-1), 127.8, 128.4, 128.5, 138.1. HRMS (CI) calcd for C14H24NO4
(M + NH4+) 270.1705; found 270.1695.
anhydrous Na2SO4. The solvents were removed in vacuo, and the
residue was purified by flash chromatography on silica gel (eluent:
hexanes:ethyl acetate, 5:1 to 1:1) to give the product 21 (83 mg, 44%
yield based on the converted alcohol) as a colorless oil, together with
Methyl 2-O-Benzyl-4-fluoro-3,4,6-trideoxy-R-D-ribo-hexopyrano-
side (18). To a solution of benzyl ether 17 (2.38 g, 9.4 mmol) in dry
CH2Cl2 (100 mL) was added 1.6 mL of (diethylamino)sulfur trifluoride
(DAST) dropwise at 0 °C. After being stirred for 1 h at room
temperature, the mixture was diluted with CH2Cl2, washed with
saturated aqueous NaHCO3, and dried over anhydrous Na2SO4. The
solvent was removed in vacuo, and the residue was purified by flash
chromatography on silica gel (eluent: hexanes:ethyl acetate, 10:1 to
5:1) to give the 4-fluorosugar 18 (0.85 g, 35% yield) as a colorless oil.
1H NMR (CDCl3, 300 MHz) δ 1.24 (3H, dd, J ) 6.3, 0.9 Hz, 5-Me),
2.01 (1H, m, 3-Hax), 2.31 (1H, m, 3-Heq), 3.42 (3H, s, OMe), 3.50 (1H,
m, 2-H), 3.71 (1H, m, 5-H), 4.02 (1H, dddd, J ) 48.6, 11.1, 9.3, 4.8
Hz, 4-H), 4.55 and 4.63 (2H, ABq, J ) 12.6 Hz, benzylic-Hs), 4.60
(1H, d, J ) 3.9 Hz, 1-H), 7.33 (5H, m, PhHs). 13C NMR (CDCl3, 75
MHz) δ 17.1 (C-6), 30.9 (d, J ) 19.1 Hz, C-3), 55.0 (OMe), 65.8 (d,
J ) 24.4 Hz, C-5), 71.2 (benzylic-C), 73.4 (d, J ) 10.9 Hz, C-2), 90.3
(d, J ) 180.2 Hz, C-4), 96.9 (d, J ) 1.6 Hz, C-1), 127.8, 127.9, 128.5,
137.9. 19F NMR (CDCl3, 188 MHz) δ -185.3 (ddd, J ) 48.7, 10.0,
4.5 Hz). HRMS (CI) calcd for C14H23FNO3 (M + NH4+) 272.1662;
found 272.1647.
Methyl 4-Fluoro-3,4,6-trideoxy-R-D-ribo-hexopyranoside (19). A
solution of compound 18 (0.85 g, 3.3 mmol) in methanol (40 mL)
containing 10% Pd/C catalyst (100 mg) was hydrogenated for 2 h under
a H2 atmosphere. The mixture was filtered through Celite, concentrated,
and coevaporated with benzene (20 mL) to give the product 19 (0.52
g, 93% yield) as a colorless oil that was used without further
purification. 1H NMR (CDCl3, 300 MHz) δ 1.26 (3H, dd, J ) 6.3, 1.5
Hz, 5-Me), 1.81 (1H, m, 3-Hax), 1.99 (1H, br s, OH), 2.35 (1H, m,
3-Heq), 3.44 (3H, s, OMe), 3.67 (2H, m, 2-H, 5-H), 4.05 (1H, dddd, J
) 48.9, 11.1, 9.3, 4.8 Hz, 4-H), 4.58 (1H, dd, J ) 4.2, 3.9 Hz, 1-H).
13C NMR (CDCl3, 75 MHz) δ 17.1 (C-6), 34.5 (d, J ) 18.5 Hz, C-3),
55.2 (OMe), 65.9 (d, J ) 24.9 Hz, C-5), 67.3 (d, J ) 11.1 Hz, C-2),
90.0 (d, J ) 180.4 Hz, C-4), 98.2 (d, J ) 1.6 Hz, C-1).19F NMR (CDCl3,
282 MHz) δ -195.3 (m). HRMS (CI) calcd for C7H17FNO3 (M +
NH4+) 182.1192; found 182.1199.
1,2-Di-O-Acetyl-4-fluoro-3,4,6-trideoxy-R-D-ribo-hexopyranose (20).
To a solution of 19 (0.52 g, 3.1 mmol) in acetic anhydride (5 mL) was
added 4 drops of concentrated sulfuric acid at 0 °C. After stirring at
room temperature for 12 h, the mixture was diluted with ethyl acetate
and H2O, neutralized with NaHCO3, washed with brine, and dried over
anhydrous Na2SO4. The solvents were removed by rotary evaporation,
and the residue was chromatographed on silica gel (eluent: hexanes:
ethyl acetate, 10:1 to 5:1) to afford the diacetyl ester 20 (0.53 g, 72%
yield, R/â > 9/1) as a colorless oil. Analytical data for the major isomer
were as follows.1H NMR (CDCl3, 300 MHz) δ 1.25 (3H, d, J ) 6.3
Hz, 5-Me), 2.00 (3H, s, OAc), 2.02 (1H, m, 3-Hax), 2.13 (3H, s, OAc),
2.36 (1H, m, 3-Heq), 3.82 (1H, m, 5-H), 4.17 (1H, dddd, J ) 48.0,
11.1, 9.3, 4.8 Hz, 4-H), 4.90 (1H, m, 2-H), 6.10 (1H, dd, J ) 3.9, 3.6
Hz, 1-H). 13C NMR (CDCl3, 75 MHz) δ 17.2 (C-6), 20.7 (OAc), 21.0
(OAc), 30.2 (d, J ) 20.6 Hz, C-3), 66.8 (d, J ) 12.1 Hz, C-2), 68.4
(d, J ) 24.9 Hz, C-5), 88.2 (d, J ) 1.6 Hz, C-1), 89.1 (d, J ) 180.5
Hz, C-4), 169.2 (CdO), 169.8 (CdO). 19F NMR (CDCl3, 282 MHz) δ
-196.2 (ddd, J ) 48.2, 9.9, 4.5 Hz). HRMS (CI) calcd for C10H19-
FNO5 (M + NH4+) 252.1247; found 252.1241.
Dibenzyl 2-O-Acetyl-4-fluoro-3,4,6-trideoxy-R-D-ribo-hexopyranyl
Phosphate (21). A mixture of diacetyl ester 20 (0.26 g, 1.1 mmol)
and tributyltin methoxide (0.4 mL) in dry CH2Cl2 (15 mL) was refluxed
for 56 h under N2. The mixture was concentrated in vacuo and
chromatographed on silica gel (eluent: hexanes:ethyl acetate, 5:1 to
2:1) to afford the C-1 deprotected sugar (0.10 g, 86% yield based on
converted starting material) along with recovered diacetyl ester 20 (0.12
g). The C-1 deprotected sugar (0.10 g, 0.52 mmol) was dissolved in
dry THF (4 mL) at -78 °C. To this solution was added n-BuLi in
hexanes (1.6 M, 0.4 mL) dropwise. After stirring for 20 min, a solution
of dibenzyl phosphorochloridate (prepared from 1 mmol of dibenzyl
phosphite and 1 mmol of N-chlorosuccinimide in benzene) in dry THF
(2 mL) was added. The resulting solution was stirred at -78 °C for 30
min, and then at 0 °C for 2 h. The mixture was concentrated at room
temperature, diluted with ether, washed with H2O, and dried over
1
the C-1 deprotected sugar (21 mg). H NMR (CDCl3, 300 MHz) δ
1.20 (3H, dd, J ) 6.3, 0.9 Hz, 5-Me), 1.92 (3H, s, OAc), 2.01 (1H, m,
3-Hax), 2.34 (1H, m, 3-Heq), 3.85 (1H, m, 5-H), 4.15 (1H, dddd, J )
48.3, 11.4, 9.9, 5.1 Hz, 4-H), 4.79-4.87 (1H, m, 2-H), 5.10 (4H, m,
2× PhHs), 5.72 (1H, m, 1-H), 7.36 (10H, m, PhHs). 13C NMR (CDCl3,
75 MHz) δ 16.9 (C-6), 29.6 (d, J ) 20.6 Hz, C-3), 67.4 (dd, J ) 12.4,
7.9 Hz, C-2), 68.0 (d, J ) 24.9 Hz, C-5), 69.4 (dd, J ) 5.5, 4.2 Hz,
benzylic-C), 88.9 (d, J ) 181.0 Hz, C-4), 93.2 (dd, J ) 5.8, 1.8 Hz,
C-1), 127.8, 127.9, 128.6, 128.7, 135.5, 135.6, 169.9.19F NMR (CDCl3,
282 MHz) δ -186.6 (dd, J ) 48.1, 4.9 Hz). 31P NMR (CDCl3, 121
MHz) δ -1.5. HRMS (FAB) calcd for C22H27FNO7P (M + H+)
453.1502; found 453.1490.
CDP-4-deoxy-4-fluoro-D-paratose (14). A suspension of 21 (103
mg) and 10% Pd/C catalyst (40 mg) in a 1:1 MeOH/EtOAc solution
(10 mL) was stirred under a H2 atmosphere at room temperature for
20 min. The mixture was filtered through Celite, and the filtrate was
neutralized with 3 drops of triethylamine and dried under reduced
pressure to give a colorless syrupy residue. The residue was stirred
with 2 N LiOH solution (10 mL) overnight and then with Dowex-
50W resin (H+ form) for 30 min. The resin was removed by filtration,
and the aqueous solution was neutralized with triethylamine and
lyophilized to give the coupling precursor (112 mg) as a white
amorphous solid. A mixture of the coupling precursor (67 mg) and
cytidine 5′-monophosphomorpholidate (274 mg) was coevaporated with
dry pyridine (3 × 1.5 mL) to form a white powder, to which was added
dry pyridine (2.0 mL) and 1H-tetrazole (48 mg). After the mixture was
stirred for 40 h at room temperature, the solvents were removed under
vacuum and the residue was dissolved in H2O. Purification was
accomplished with a Bio-Rad P2 column (2 cm × 1.2 m) using 50
mM NH4HCO3 as the mobile phase. The desired fractions were detected
by UV absorption at 267 nm and lyophilized to give the coupling
product 14 (59 mg, 54% yield) as a white powder.1H NMR (2H2O,
300 MHz) δ 1.02 (3H, d, J ) 7.2 Hz, 5-Me), 1.65 (1H, m, 3-Hax), 2.07
(1H, m, 3-Heq), 3.58 (1H, m, 2-H), 3.74 (1H, m, 5-H), 3.91-4.15 (6H,
m, 4-H, 2′-H, 3′-H, 4′-H, 5′-Hs), 5.23 (1H, m, 1-H), 5.72 (1H, d, J )
3.6 Hz, 1′-H), 5.90 (1H, d, J ) 7.5 Hz, 5′′-H), 7.77 (1H, d, J ) 7.8
Hz, 6′′-H). 13C NMR (2H2O, 75 MHz) δ 16.1, 32.1 (d, J ) 18.8 Hz),
64.2 (d, J ) 5.4 Hz), 67.1 (d, J ) 5.4 Hz), 68.4, 74.2, 82.3 (d, J ) 9.6
Hz), 89.5, 89.8 (d, J ) 176 Hz), 94.0 (d, J ) 5.4 Hz), 96.4, 141.2,
157.5, 160.2, 166.1.19F NMR (2H2O, 282 MHz) δ -185.3 (dd, J )
48.2, 5.6 Hz). 31P NMR (2H2O, 121 MHz) δ -10.5 (d, J ) 21.4 Hz),
-12.1 (d, J ) 21.4 Hz). HRMS (ESI) calcd for C15H23FN3P2O13 (M -
H-) 534.0690; found 534.0688.
Investigation of CDP-4-deoxy-4-fluoro-D-paratose (14) as a
Substrate. CDP-4-deoxy-4-fluoro-D-paratose (14, 7.4 nmol) was mixed
with 0.1 mg of CDP-D-tyvelose 2-epimerase in 100 µL of 20 mM Tris-
HCl buffer (pH 7.5). After incubation at room temperature for 1 h, the
sample was analyzed by HPLC using a SAX column as described under
“Enzyme Assay”. A large incubation was performed where 37 µmol
of 14 and 4.5 mg of CDP-D-tyvelose 2-epimerase were incubated in
1.3 mL of 20 mM Tris-HCl buffer (pH 7.5) for approximately 8 h at
room temperature. The product, CDP-4-deoxy-4-fluoro-D-tyvelose, was
purified by FPLC as described under “Production and Characterization
1
1
of CDP-D-tyvelose” and analyzed by H NMR. H NMR (2H2O, 300
MHz) δ 0.94 (3H, d, J ) 6.3 Hz, 5-Me), 1.71 (1H, dddd, J ) 3.0,
13.2, 13.2, 13.2 Hz, 3-Hax), 1.87 (1H, m, 3-Heq), 3.73 (2H, m, 2-H,
5-H), 3.84-4.02 (5H, m, 2′-H, 3′-H, 4′-H, 5′-Hs), 4.18 (1H, dddd, J )
4.8, 9.3, 9.3, 49 Hz, 4-H), 4.96 (1H, m, 1-H), 5.65 (1H, d, J ) 3.9 Hz,
1′-H), 5.80 (1H, d, J ) 7.5 Hz, 5′′-H), 7.62 (1H, d, J ) 7.5 Hz, 6′′-H).
13C NMR (2H2O, 75 MHz) δ 16.2, 30.3 (d, J ) 20.4 Hz), 64.5 (d, J )
5.6 Hz), 68.2 (d, J ) 25 Hz), 68.9, 73.9, 82.3 (d, J ) 9.3 Hz), 89.0 (d,
J ) 171 Hz), 89.2, 94.7 (d, J ) 6.1 Hz), 96.4, 141.2, 157, 160.4, 165.9.
19F NMR (2H2O, 282 MHz) δ -190.4 (dd, J ) 5, 48 Hz). 31P NMR
(2H2O, 121 MHz) δ -10.8 (d, J ) 50 Hz), -13.0 (d, J ) 50 Hz).
HRMS (FAB): calcd for C15H23FN3O13P2 534.0690; found 534.0703.
Metal Analysis of CDP-D-tyvelose 2-Epimerase by Inductively
Coupled Plasma (ICP) Spectroscopy. CDP-D-tyvelose 2-epimerase