114
R. Hardre´, L. Salmon / Carbohydrate Research 318 (1999) 110–115
4 H, H-3,4,5,5%); 31P NMR (D2O): l 4.72;
droxamic acid and HONH3+), and 1093 (OH
1
and PO4−) cm−1; H NMR (D2O): l 4.46 (br
HR-EIMS (negative mode, TMS derivative):
−
s, 1 H, H-2), 3.71–3.99 (m, 4 H, H-3,4,5,5’);
Anal. Calcd for C23H60NO8PSi6 [M]
677.2672, Found: 677.2687.
:
13
31P NMR (D2O): l 3.82; C NMR and high-
resolution MS data: see Ref. [16].
D
-Arabinonhydrazide-5-phosphate, monohy-
D
-Arabinono-1,4-lactone
5-(dihydrogeno-
drazinium salt (4).—In a round-bottom flask
placed under argon, equipped with a septum
phosphate) (5) [16].—Compound 2, barium
salt (50 mg, 0.11 mmol) obtained from either
G6P [16] or F6P [17] was first solubilized in
water (6 mL) by stirring with a Dowex®
50WX8-100 cation-exchange resin (H+ form,
1 g). The resin was then removed by filtration
on a glass funnel, and the solution was ad-
sorbed on a Dowex® 50WX8-100 cation-ex-
change resin column (H+ form, 3 g, h=14.5
cm, d=1 cm). Following elution of the
product with water until the eluent reached
neutral pH (approx. 60 mL), removal of most
of the solvent under reduced pressure, and
finally, lyophilization, the title compound was
obtained as a colorless syrup in quantitative
yield (26 mg, 0.11 mmol): [h]2D8 +28.6° (c 3.2,
water); FTIR (KBr) w 3382 (alcohol), 2919
(CH), 1778 (lactone), 1424–1384 (lactone),
and containing
D
-arabinono-1,4-lactone 5-
phosphate 5 (91 mg, 0.40 mmol) in anhydrous
MeOH (5 mL), was added dropwise and
through a syringe hydrazine, monohydrate (70
mL, 1.44 mmol). A white precipitate appeared
immediately. The reaction mixture was stirred
at rt for 1 h. After removal of the solvent
under reduced pressure, the crude solid was
purified by size-exclusion chromatography
(water) on a Bio-Gel® P-2 resin from Bio-Rad
(200–400 mesh, 25 g, h=95 cm, d=1 cm),
and subsequently lyophilized to give the title
compound 4 (76 mg, 78%) as a white solid:
+19.6° (c 1.6, water); FTIR (KBr) w 3323
(OH), 1631 (hydrazide), and 1097 (OH and
PO4−) cm−1; H NMR (D2O): l 4.36 (d, 1 H,
1
J2,3 1.5 Hz, H-2), 3.89–3.62 (m, 4 H, H-
3,4,5,5%); 31P NMR (D2O): l 4.40; HR-EIMS
(negative mode, TMS derivative): Anal. Calcd
and 1130–1067 (OH and PO4−) cm−1; H
1
NMR (D2O): l 4.48 (d, 1 H, J2,3 8.8 Hz, H-2),
4.19 (t, 1 H, J3,4 8.8 Hz, H-3), 4.32 (ddd, 1 H,
J4,5 4.4, J4,5% 2.4 Hz, H-4), 4.00 (ddd, 1 H, J5,5%
−12.7, J5,P 6.8 Hz, H-5), 4.16 (ddd, 1 H, J5%,P
5.8 Hz, H-5%); 31P NMR (D2O): l 3.34; 13C
NMR and high-resolution MS data: see Ref.
[16].
for C20H53N2O8PSi5 [M] −: 620.2386, Found:
620.2384.
Enzyme kinetic assays.—Enzymes, b-nicoti-
namide adenine dinucleotide phosphate
(NADP, sodium salt), F6P and G6P (dis-
odium salts) were purchased from Sigma
Chemical Company. Yeast PGI was assayed
at 30 °C spectrophotometrically (u=340 nm)
in the direction from F6P to G6P by coupling
D
-Arabinonamide-5-phosphate, diammonium
salt (3).—In a round-bottom flask placed un-
der argon, containing -arabinolactone-5-
D
phosphate 5 (88 mg, 0.39 mmol) and cooled in
a dry-ice/isopropanol bath (−78 °C), approx-
imately 10 mL of anhyd liquid ammonia were
condensed. The reaction mixture was left to
proceed for 10 min. Then, the flask was taken
off the dry-ice/isopropanol bath in order for
the liquid ammonia to evaporate at room
temperature. Complete removal of liquid am-
monia was achieved with a stream of argon.
Following drying over P2O5 under reduced
pressure for 4 days, the title compound 3 (114
mg, quantitative yield) was obtained as a
white solid: −8.3° (c 1.1, water); FTIR (KBr)
w 3201 (OH), 1671 (amide), 1401 (amide), and
to yeast -glucose-6-phosphate dehydrogenase
D
(PGDH) and NADP [25,26]. The reaction
mixture (1 mL) had the following composition
(final concentrations): 50 mM TRIS, pH 8.0;
0.1–0.5 mM F6P; 1 mM EDTA; 0.4 mM
NADP; 0.0–0.2 mM selected inhibitor. To
ensure valid assay conditions, several precau-
tions were taken. Sufficient PGDH (0.6 U)1
was added and the reaction mixture was al-
lowed to pre-incubate for 6–8 min. The reac-
tion was then initiated by the addition of the
PGI (0.0051 U). The ratio of activities in term
of units of PGDH to PGI was kept at 120:1.
1
1097 (OH and PO4−) cm−1; H NMR (D2O):
1 1 unit (U) is defined as that quantity of enzyme which will
convert 1 mmol of substrate to product per min at 30 °C.
l 4.24 (d, 1 H, J2,3 1.5 Hz, H-2), 3.80–3.56 (m,