1228
PISHCHUGIN, TULEBERDIEV
water–alcohol buffer systems and kept at a given
D
temperature for 30 min at pH ~7. The reaction was
considered to start in the moment when HCl or NaOH
was added. Kinetic measurements were performed in
temperature-controlled cells with a layer thickness of
1.008 mm. Taking into account that the UV spectra of
pyridoxal hydrochloride and pyridoxal 5'-phosphate
depend on solvent and medium pH, as reference we
used solvents or solutions of pyridoxal hydrochloride
or pyridoxal 5'-phosphate in conditions corresponding
to the conditions of hydrolysis of the Schiff bases.
Such approach excluded the contribution of the optical
density of the final compounds into the optical density
of the reaction mixture. To account for the
precipitation of keto acid salts, attendant in chemical
transformations of pyridoxal or pyridoxal 5'-phosphate
condensation products with D,L-triptophan and L-
glutamic acid in alkaline media, simultaneously in the
same thermostat under the same conditions we kept the
solutions of the Schiff bases and measured the time of
precipitation initiation.
1.2
1.0
0.8
0.6
0.4
0.2
1
2
80 240 400 560 720 880
τ, min
Fig. 3. Kinetics of (1) pyridoxal and (2) pyridoxal 5'-
phosphate condensation with D,L-triptophan (70% water-
alcohol buffer, pH 7.1, 50°С, λmax 430 nm).
This scheme is evidenced by the elemental analyses
and UV and IR spectra of the formed precipitates. Our
results are consistent with the conclusion of Dunathan
[9] that the bond cleaved by a pyridoxal 5'-phosphate–
dependent enzyme should be located in a plane
orthogonal to the plane of the imine π system of the
coenzyme substrate. Such orientation minimizes the
energy of the transition state, because it ensures the
best σ–π overlap of the cleaved bond in the conjugated
coenzyme imine π system.
The rate constants of hydrolysis of pyridoxal and
pyridoxal-5'-phosphate condensation products with
amino acids were calculated from the calibration
straight lines by the first-order reversible and
irreversible reaction equations [10]. The Schiff bases
and their chemical transformation products were
identified by elemental analysis, UV and IR
spectroscopy, and column chromatography. The Schiff
bases were synthesized by a common procedure [7].
As starting materials we used pyridoxal hydrochloride
and pyridoxal 5'-phosphate from Ferak, Berlin and
amino acids from Reanal. Equimolar amounts of
pyridoxal hydrochloride or pyridoxal 5'-phosphate and
amino acid were heated at 60–70°С for 15–30 min,
and the resulting compounds were isolated and
recrystallized. The synthesis of pyridoxalidene glycine
and sodium β-(3-indolyl)-α-ketopropanoate were
described in [7].
Thus, the results of our investigation show that
pyridoxal or pyridoxal 5'-phosphate condensation
products with amino acids are the most stable at pHs
close to neutral. In acid media, they decompose due
to hydrolysis into the initial components. In alkaline
media, cleavage of the α-hydrogen atom in the amino
acid fragment of the Schiff base, formation of the
quinoid structure, and its subsequent hydrolysis occur,
leading to pyridoxamine or pyridoxamine 5'-phosphate
and keto acid.
Thus, we obtained the first experimental evidence
showing that the phosphate group in pyridoxal 5'-
phosphate condensation products with amino acids can
catalyze α-hydrogen cleavage and transfer of the
Schiff base into the quinoid structure whose hydro-
lysis accelerates formation of pyridoxamine 5'-
phosphate and keto acids.
Pyridoxalidene D,L-triptophan. To a mixture of
0.103 g of pyridoxal hydrochloride and 0.102 g of D,L-
triptophan, 35–40 ml of 90% ethanol was added with
vigorous stirring at pH 6.7–7.0 and 25°С, and the
reaction mixture was kept until the reagents dissolved
completely. Therewith, the solution got intensely
yellow. The solution was left overnight in a freezer.
The yellow precipitate that formed was washed with a
little water and then with ethanol. Yield 72–75%. IR
spectrum (KBr), ν, cm–1: 1635 (С=N) , 1615–1627
(COO–). UV spectrum, λmax, nm: 355, 430. Found, %:
EXPERIMENTAL
The kinetics of hydrolysis of pyridoxal and
pyridoxal 5'-phosphate condensation products with
amino acids were studied using a SpectroMOM-204
spectrophotometer. The reaction mixture was
thermostated in a UН–8 thermostat with an accuracy
of 0.1°С . Samples of Schiff bases were dissolved in
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 78 No. 6 2008