Synthetic Utility of Yeast Hexokinase
J . Org. Chem., Vol. 62, No. 2, 1997 335
from Biozyme Laboratories and were used without additional
purification. Compounds 31,40 32,41 and 3442 were made
according to previously reported procedures or slight modifica-
tions thereof.43,44 1H, 13C, and 31P NMR spectra were acquired
at nominal resonance frequencies of 250 and 101 MHz,
respectively, using D2O as the solvent. FAB-MS data were
obtained using Cs+ (20 eV) as the ionizing beam and glycerol/
NaI as the matrix. Elemental analyses were performed by
Robertson Microlit Laboratories, Inc. TLC was performed on
6.7 cm × 2 cm normal-phase TLC plates (250 µm sorbent
thickness) from either Analtech (Silica Gel HL) or Whatman
(K6F silica gel 60 Å). Silica gel chromatography was per-
formed on Merck grade 9385, 230-400 mesh, 60-Å silica gel.
TLC Assa y for Hexok in a se Activity. In a final volume
of 1.00 mL of 0.10 M sodium phosphate buffer, pH 7.6, at rt,
were combined 50 mM glucose or unnatural substrate, 5.0 mM
ATP, 5.0 mM MgCl2, and 1.0 or 10 U of hexokinase. Hexoki-
nase (20 or 200 µL of a 50 U mL-1 solution) was added last to
initiate the reaction. Control reactions containing all of the
components but hexokinase in the same final volume were also
run. Reactions were monitored periodically by TLC (9.5 mM
tetrabutylammonium hydroxide in 80% aqueous acetonitrile,
anisaldehyde stain) for the presence of sugar phosphate.
Under these conditions, Rf values of glucose, ATP, and glucose
6-phosphate were 0.53, 0.16, and 0.30, respectively.
Sp ectr op h otom etr ic Assa y for Hexok in a se Activity.
To 1.465 mL of 0.10 M sodium phosphate buffer, pH 7.6,
containing 14.4 U of pyruvate kinase, 2.15 U of lactate
dehydrogenase, 50 mM substrate, 2.36 mM ATP, 3.30 mM
MgCl2, 13.2 mM KCl, and 0.393 mM NADH, at 25 °C, was
added 50 µL of a 0.75 or 7.5 U mL-1 solution of hexokinase.
The absorbance at 334 nm was recorded continuously for about
10 min, and the activity of hexokinase was calculated using
ꢀ334 ) 6.18 L mmol-1 cm-1 as the molar absorptivity of NADH.
Gen er a l P r oced u r e for P r ep a r a tive P h osp h or yla tion s.
In a 125-mL Erlenmeyer flask equipped with a stir bar were
combined substrate (4.3 mmol), 17 mg (0.031 mmol) of ATP,
51 mg (0.25 mmol) of MgCl2‚6H2O, 37 mg (0.50 mmol) of KCl,
23 mg (0.15 mmol) of dithiothreitol, 6.5 mg (0.10 mmol) of
NaN3, and 20 mL of deoxygenated, deionized water. The pH
of the solution was adjusted to 7.6 with aqueous potassium
hydroxide, and the solution was sparged with nitrogen. Hexo-
kinase (45-60 U, determined with the actual compound being
phosphorylated) and pyruvate kinase (45-60 U) were added.
To this solution (final volume 25 mL), under an inert atmo-
sphere, was added by syringe pump 25 mL of a deoxygenated
solution of 4.7 mmol of phosphoenolpyruvate,45 pH 7.6. Upon
completion of the addition of phosphoenolpyruvate, the reac-
tion was assayed for phosphoenolpyruvate and pyruvate.
After completion of the reaction, the solution was adjusted
to pH 9.0 using aqueous potassium hydroxide, and barium
acetate (1.8 g, 6.85 mmol) dissolved in 4 mL of water was
added. The mixture was stirred for 20 min and then filtered
to remove barium phosphate. Residual barium was removed
by adding 30-35 mmolar equiv of Dowex-50W (H+), stirring,
and filtering. The solution was adjusted to pH 9.0 with
aqueous potassium hydroxide and brought to the brink of
precipitation by the addition of methanol. The solution was
applied to a 150-mL column of Dowex-1×8-100 (HCO3-),
which was then rinsed with 200-300 mL of 50% aqueous
methanol, eluted with 3 L of 65 mM ammonium bicarbonate
in 50% aqueous ethanol to remove carboxylate anions, and
then eluted with 3 L of 200 mM aqueous ammonium bicarbon-
ate. The latter eluant was concentrated by rotary evaporation
at 40 °C to yield sugar phosphate as its diammonium salt.
Aminosugar phosphates were recovered as their monoammo-
nium salts.
For storage or FAB-MS analysis, sugar phosphates were
converted to their monosodium or bis(cyclohexylammonium)
salts. Monosodium salts were formed by stirring sugar
phosphates in water with excess Dowex-50W (H+), filtering,
raising the pH of the solution to 4.2 with aqueous sodium
hydroxide, and removing the solvent by rotary evaporation.
Bis(cyclohexylammonium) salts were formed by dissolving
sugar phosphates in water with an excess of Dowex-50W (H+),
stirring, filtering, adding 2 equiv of cyclohexylamine, and
removing the solvent by rotary evaporation. The bis(cyclo-
hexylammonium) salts were recrystallized from water-2-
propanol.
D-Glu cose 6-P h osp h a te (47). Phosphorylation of 1 ac-
cording to the general procedure gave 1.04 g (80%) of 47
diammonium salt: 1H NMR δ 5.28 (d, J ) 3.8 Hz, 0.3H, H-1R),
4.70 (d, J ) 8.0 Hz, 0.7H, H-1â), 4.08-3.92 (m, 2H, H-6,6′),
3.81-3.51 (m, 3.3H), 3.33 (t, J ) 9.6 Hz, 0.7H), in agreement
with authentic material purchased from Sigma.
2-Deoxy-D-glu cose 6-P h osp h a te (48). Phosphorylation of
2 according to the general procedure yielded 0.95 g (79%) of
48 bis(cyclohexylammonium) salt that was a 1:1 mixture of R
and â anomers: 1H NMR δ 5.37 (d, J ) 3.2 Hz, 0.5H), 4.94
(dd, J ) 1.8, 9.8 Hz, 0.5H), 4.11-3.85 (m, 3H), 3.73 (m, 0.5H),
3.55 and 3.47 (2 t, J ) 9.7 Hz, 0.5H each), 3.44 (m, 0.5H), 3.15
(m, 2H), 2.27 (ddd, J ) 1.9, 5.0, 12 Hz, H-2eqâ), 2.14 (dd, J )
5.1, 13 Hz, H-2eqR), 1.98 (m, 4H), 1.80 (m, 4H), 1.74 (dt, J )
3.2, 13 Hz, 0.5H, H-2axR), 1.54 (dt, J ) 10, 12 Hz, 0.5H,
H-2axâ), 1.34 (m, 12H), in agreement with authentic material
(sodium salt) purchased from Sigma; 31P NMR δ 4.31.
Meth yl r-D-Glu cop yr a n osid e 6-P h osp h a te (49). Phos-
phorylation of 5 according to the general procedure yielded
0.65 g of 49 monosodium salt as a white solid: mp 138-140
°C dec; 1H NMR δ 4.81 (d, J ) 3.6 Hz, 1H, H-1), 4.09 (m, 2H,
H-6,6′), 3.76 (m, 1H, H-5), 3.65 (t, J ) 9 Hz, 1H), 3.59 (dd, J
) 3.5, 9.4 Hz, 1H, H-2), 3.52 (t, J ) 9.4 Hz, 1H), 3.43 (s, 3H,
OCH3); 31P NMR δ 3.94; IR (KBr) 3464, 1637, 1471, 1404, 1366,
1263, 1203, 1049, 931 cm-1; FAB-MS m/z 319 (M + Na+), 297
(M + H+), 275 (free acid + H+). Anal. Calcd for C7H14O9-
NaP‚H2O: C, 26.76; H, 5.13; P, 9.86. Found: C, 26.94; H, 5.34;
P, 10.00.
Meth yl r-D-Ma n n op yr a n osid e 6-P h osp h a te (50). Phos-
phorylation of 8 according to the general procedure yielded
0.97 g (73%) of 50 monosodium salt as a white solid: mp 145-
1
147 °C; H NMR δ 4.77 (br s, 1H, H-1), 4.05 (m, 2H, H-6,6′),
3.94 (dd, J ) 1.6, 3.0 Hz, 1H, H-2), 3.86-3.80 (m, 2H, H-4, 3),
3.65 (m, 1H, H-5), 3.42 (s, 3H, OCH3); 31P NMR δ 4.58; IR
(KBr) 3526, 2917, 1448, 1389, 1353, 1323, 1197, 1022, 970,
930 cm-1; HRMS calcd for C7H15O9NaP (M + H+) 297.0348,
found 297.0351.
D-Ma n n ose 6-P h osp h a te (51). Phosphorylation of 9 ac-
cording to the general procedure yielded 1.34 g (99%) of 51
monosodium salt as a white solid: 1H NMR δ 5.19 (d, J ) 1.58
Hz, 0.6H, H-1R), 4.92 (d, J ) 1.02 Hz, 0.4H, H-1â), 4.02-3.99
(m, 2H, H-6,6′), 3.94 (m, 1H, H-2), 3.86 (m, 2H), 3.72 (m, 1H),
in agreement with the literature;35 31P NMR δ 4.90; HRMS
calcd for C6H13NaO9P (M + H+) 283.0205, found 283.0195.
2-Am in o-2-d eoxy-D-glu cose 6-P h osp h a te (52). Phos-
phorylation of 16 according to the general procedure yielded
1.22 g (100%) of 5230,46-48 monoammonium salt: 1H NMR δ
5.46 (d, J ) 3.6 Hz, 0.7H, H-1R), 4.46 (d, J ) 8.5 Hz, 0.3H,
H-1â), 4.17-4.04 (m, 2H, H-6,6′), 4.01 (m, 0.7H, H-5R), 3.92
(dd, J ) 9.3, 10.5 Hz, 0.7H, H-3R), 3.71 (dd, J ) 8.5, 10.5, 0.3H,
H-3â), 3.66-3.57 (m, 1.3H, H-4R,4â,5â), 3.34 (dd, J ) 3.6, 10.5
Hz, 0.7H, H-2R), 3.01 (dd, J ) 8.5, 10.5 Hz, 0.3H, H-2â), in
agreement with authentic material (sodium salt) purchased
from Sigma; 31P NMR δ 4.49; HRMS calcd for C6H15NO8P (M
+ H+) 260.0529, found 260.0535.
(40) Foster, A. B.; Stacey, M.; Vardheim, S. V. Acta Chem. Scand.
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(42) Chenault, H. K.; Mandes, R. F. Tetrahedron, in press.
(43) Compound 31 was prepared by deacetylation of 3,4,6-tri-O-
acetyl-D-glucal, followed by catalytic hydrogenation.
(44) Deoxygenation (with double-bond migration) of 3,4,6-tri-O-
acetyl-D-glucal with LiClO4-Et3SiH (Wustrow, D. J .; Smith, W. J ., III;
Wise, L. D. Tetrahedron Lett. 1994, 35, 61-64) was followed by
catalytic hydrogenation and deacetylation to give 32.
(45) Hirschbein, B. L.; Mazenod, F. P.; Whitesides, G. M. J . Org.
Chem. 1982, 47, 3765-3766.
(46) Brown, D. H. Methods Enzymol. 1957, 3, 158-162.
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(48) J ourdian, G. W.; Roseman, S. Biochem. Prep. 1962, 9, 44-47.