Chemical transformations of the condensation products of pyridoxal with L-α-alanine and D-α-alanine

Article abstract of DOI:10.1134/S1070363209010174

The kinetics and mechanism of the reactions of pyridoxal with L- and D-α-alanine were studied. Under comparable conditions, the condensation of L- and D-α-alanines with pyridoxal includes three kinetically different steps. The first fast step is addition

Full text of DOI:10.1134/S1070363209010174

ISSN 1070-3632, Russian Journal of General Chemistry, 2009, Vol. 79, No. 1, pp. 117–120. © Pleiades Publishing, Ltd., 2009.
Original Russian Text © F.V. Pishchugin, I.T. Tuleberdiev, 2009, published in Zhurnal Obshchei Khimii, 2009, Vol. 79, No. 1, pp. 120–123.
Chemical Transformations of the Condensation Products
of Pyridoxal with L-α-Alanine and D-α-Alanine
F. V. Pishchugin and I. T. Tuleberdiev
Institute of Chemistry and Chemical Technology, National Academy of Sciences of Kirgizia,
pr. Chui 267, Bishkek, 720071 Kirgiziya
e-mail: pishugin@rambler.ru
Received October 23, 2007
Abstract—The kinetics and mechanism of the reactions of pyridoxal with L- and D-α-alanine were studied.
Under comparable conditions, the condensation of L- and D-α-alanines with pyridoxal includes three kinetically
different steps. The first fast step is addition of the amino acid to pyridoxal with formation of the corresponding
amino alcohol, the second (slower) step is dehydration of the amino alcohol to give Schiff base, and the third
(
very slow) step is elimination of α-hydrogen atom from the L-α-amino acid fragment or decarboxylation of the
D-α-amino acid fragment, followed by isomerization of the Schiff base to quinoid structure whose subsequent
hydrolysis yields pyridoxamine and pyruvic acid or acetaldehyde, respectively. A scheme was proposed for
chemical transformations of the pyridoxal condensation products with L- and D-α-alanines.
DOI: 10.1134/S1070363209010174
Pyridoxal and pyridoxal-5′-phosphate (PLP) are
coenzymes in a large number of enzymatic systems
that catalyze biochemical transformations of amino
acids and amines, such as transamination, elimination,
decarboxylation, side chain cleavage in amino acids,
etc. Studies on the mechanism of action of the above
enzymes and their models have been reported in some
publications [1, 2]. However, taking into account
complexity of such systems and high rates and
sometimes ambiguous results of enzymatic processes,
problems related to the kinetics and mechanism of
chemical transformations of amino acid still remain to
be solved.
In some studies, para-nitrosalicylaldehyde was
proposed as coenzyme model [1]. Quantum-chemical
optimization of the energetic and structural parameters
showed that, in contrast to para-nitrosalicylaldehyde
molecule in which the benzene ring and the carbonyl
group lie in one plane, the carbonyl group in pyridoxal
and pyridoxal-5′-phosphate molecule is turned through
an angle of 180° with respect to the pyridine ring
plane, presumably due to the presence of the hydroxy
and hydroxymethyl groups at the neighboring carbon
atoms. Therefore, the use of para-nitrosalicylaldehyde
as a model of pyridoxal seems to be inappropriate.
It was presumed [5] that the bond cleaved by PLP-
dependent enzyme in amino acid (substrate) should be
oriented orthogonally to the plane of the π-system in
the substrate–coenzyme condensation product.
According to the author, such orientation minimizes
the energy of transition state, for it ensures maximal σ–
π overlap of the bond being cleaved and the conjugated
π-system in the cofactor–imine system and the most
favorable geometry for the transformation into quinoid
state. Such enzymes catalyze decarboxylation of, e.g.,
D-alanine and elimination of hydrogen from the α-
position of L-alanine [1].
We previously [3, 4] studied the kinetics of
reactions of pyridoxal and pyridoxal-5′-phosphate with
amino acids and amino sugars and found that that the
reactions with amino acids and aminodeoxy sugars
include several steps. The first very fast step is
addition of an amino acid to coenzyme with formation
of amino alcohol. The second step is slower, and it
involves dehydration of amino alcohol to the
corresponding Schiff base. The third (slowest) step
includes elimination of structural fragments of amino
acids or aminodeoxy sugars to give quinoid structures
whose hydrolysis leads to the final products, py-
ridoxamine, keto acid or keto sugar. The rate of each
step depends on the solvent, pH, and temperature.
It was interesting to examine the kinetics of con-
densation of pyridoxal with L- and D-stereoisomers of
117

1
18
PISHCHUGIN, TULEBERDIEV
stereoisomers to pyridoxal, because of very high rate
D
of this process even at low temperature. The rate of
1
.2
.0
.8
.6
.4
.2
dehydration of the amino alcohol derived from L-α-
2
1
1
0
0
0
0
–1
alanine was slightly higher (k = 0.1646 min ) than the
2
rate of dehydration of its stereoisomer containing D-α-
–
1
3
alanine fragment (k = 0.1453 min ). Taking into
2
account that the carbonyl group in pyridoxal molecule
is turned through an angle of 180° with respect to the
pyridine fragment, addition of stereoisomeric acids
yields amino alcohols with different mutual arrange-
ments of the pyridine and amino acid fragments.
Intermediate amino alcohols are likely to be stabilized
by formation of hydrogen bonds between the OH and
NH groups. Quantum-chemical calculations performed
in the MNDO and MNDO1 approximations showed
that the steric structure and charges on the reaction
centers are more favorable for dehydration of the
amino alcohol having L-alanine fragment, as compared
to its D-alanine stereoisomer. Therefore, the rate of
dehydration of the former is higher.
4
1
0
30 50 100 200 300 400
τ, min
Variations of the optical densities at (1–3) λ 430 nm of
equimolar mixtures of 0.01 M solutions of pyridoxal with (1)
L-α-alanine, (2), D-α-alanine, and (3) ethanamine hydro-
chloride and (4) at λ 350 nm of the hydrolysis product of
quinoid structure (pyridoxamine); 90% aqueous alcoholic
acetate buffer, pH 7.15, 50°C.
α-alanine under comparable conditions, determine the
structure and energies of the initial compounds,
intermediates, and final products and charges on the
reaction centers therein by quantum-chemical methods,
isolate intermediate and final products, and elucidate
the mechanism of these transformations.
It was specifically interesting to examine the
kinetics and mechanism of the third step which could
take different pathways. The first of these is
elimination of hydrogen from the α-position and iso-
merization of intermediate Schiff base into quinoid
structure, and the subsequent hydrolysis yields pyridox-
amine and oxo acid (pyruvic acid; Scheme 1).
Analysis of the kinetic curves (see figure) and
calculation of the rate constants for all irreversible and
reversible steps showed that L-α-alanine is generally
more reactive than D-α-alanine in the condensation
with pyridoxal. Unfortunately, we failed to measure
the kinetic of the first step, addition of alanine
Scheme 1.
H
−OOC
−OOC
CH3
CH
CH3
CH
C
N
C
N
H
O
H
O
CH OH
CH OH
2
2
−Η+
H3C
H3C
+
N
H
N
H
+NH3
CH2
−O
CH OH
2
Η Ο
2
+
CH −C−COOH
3
H3C
+
N
O
H
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 79 No. 1 2009

CHEMICAL TRANSFORMATIONS OF THE CONDENSATION PRODUCTS
119
The second pathway involves decarboxylation of
the Schiff base, transformation into quinoid structure,
and hydrolysis to produce pyridoxamine and acetal-
dehyde (Scheme 2).
Scheme 2.
COO−
CH3
CH
CH3
H
C
N
N
H
O
CH
H
O
CH
CH OH
CH OH
2
2
H C
−CΟ
2
H C
3
+
3
N
N
H
H
+NH3
CH2
−O
CH OH
O
2
Η Ο
2
+
CH3
C
H C
3
H
+
N
H
As follows from the kinetic data (see figure), the
rate of the third step including elimination, formation
of quinoid structure, and hydrolysis is higher for the Schiff
In order to confirm the proposed schemes we
synthesized the corresponding Schiff bases. Under the
condensation conditions, the concentration of Schiff
bases (λ 430 nm) decreased with time in both cases,
while the concentration of pyridoxamine (λ 350 nm)
simultaneously increased (see figure). In the condensa-
tion of pyridoxal with L-α-alanine we succeeded in
isolating and identifying sodium oxoacetate by
evaporation of the solvent and treatment of the residue
with excess ethanol. In the condensation of pyridoxal
with D-α-alanine, an equimolar amount of barium
hydroxide was added to the reaction mixture after
equilibration (i.e., when the optical densities at λ 350
and 430 nm no longer changed). During the addition,
–
1
base having L-α-alanine fragment (k = 0.00363 min );
3
the corresponding rate constant for the Schiff base
–1
derived from D-alanine is k3 = 0.00173 min .
Quantum-chemical study on the structure of the
stereoisomeric Schiff bases with optimization of
energetic and geometric parameters showed that the
–
CH=N– bond in both stereoisomers deviates from the
α
pyridine ring plane. The C –H bond in the L-amino
acid residue is oriented orthogonally to the pyridine
ring plane, whereas in the Schiff base derived from
–
D-α-alanine, the COO group lies in the plane
orthogonal to the pyridine ring.
the solution turned turbid due to formation of BaCO .
3
It should be noted that addition of Ba(OH) to the
2
In keeping with the concept developed in [5–8], in
the third step the condensation product of pyridoxal
with L-α-alanine undergoes elimination of α-hydrogen
atom, transformation into quinoid form, and hydrolysis
with formation of pyridoxamine and pyruvic acid
reaction mixture obtained from D-α-alanine did not
produce turbidity. Furthermore, the pH value of the
equilibrium mixture changed from 5.6 to 5.3, which
may be related to evolution of CO as a result of decar-
2
boxylation. The decarboxylation product, N-(3-hyd-
roxy-5-hydroxymethyl-2-methylpyridin-4-ylmethylidene)
ethanamine was identified by chromatography, as well
as by independent synthesis from pyridoxal and ethan-
amine hydrochloride.
(
Scheme 1). The Schiff base having D-α-alanine
fragment undergoes decarboxylation to give quinoid
structure whose hydrolysis leads to pyridoxamine and
acetaldehyde (Scheme 2).
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 79 No. 1 2009

1
20
PISHCHUGIN, TULEBERDIEV
EXPERIMENTAL
Commercial pyridoxal hydrochloride (Ferak Berlin)
and stereoisomeric L-α-alanine and D-α-alanine (Reanal)
were used. Buffer solutions were prepared according to
standard procedures. The kinetics of the condensation
Found, %: C 41.17; H 4.78; N 9.77. C10
Calculated, %: C 42.1; H 4.92; N.84.
H N O ·HCl.
13 2 4
N-(3-Hydroxy-5-hydroxymethyl-2-methylpyridin-
-ylmethylidene)-D-α-alanine. Yield 0.096 g (64%).
4
Found, %: C 42.0; H 4.85; N 9.64. C H N O ·HCl.
1
0
13
2
4
Calculated, %: C 42.1; H 4.92; N.84. IR spectrum, ν,
were studied by spectrophotometry using
a
–
1
–
cm : 1631 (C=N); 1528, 1414 (COO ).
Spektromom-204 instrument. The reaction mixtures
were maintained at a constant temperature with an
accuracy of ±0.1°C using a UH-8 thermostat. Equi-
molar amounts of pyridoxal and amino acid were
dissolved in aqueous–alcoholic buffers, and the
solutions were kept for 30 min at a required tempera-
ture. The moment of mixing of the reactant solutions
was taken as the reaction start. Cells with a path length
of 1.008 mm were used for spectrophotometric
measurements. Taking into account that the UV
spectrum of pyridoxal hydrochloride depends on the
solvent and pH, the corresponding solvent or buffer
solution was placed into the reference cell. The
condensation rate constants were calculated by first-
and second-order equations for reversible and
irreversible reactions [9] using calibration curves. The
initial compounds and intermediate and final products
were identified by elemental analysis and UV and IR
spectoscopy (Nicolet Avatan 370 PGTS, KBr pellets).
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The Schiff bases were synthesized according to the
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-ylmethylidene)-L-α-alanine. Yield 0.11 g (73%).
4
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