Kinetic and mechanism of reactions of L-α-glutamic acid and L-Glutamine with pyridoxal

Article abstract of DOI:10.1134/S1070363214070196

The kinetics and mechanisms of condensation of pyridoxal with L-α-glutamic acid and L-glutamine were studied by UV spectroscopy and polarimetry. L-α-Glutamic acid reacts with pyridoxal to form a Schiff base whose subsequent hydrolysis gives rise to pyridoxamine and α-ketoglutaric acid. The reaction of Lglutamine with pyridoxal involves the Γ-NH ₂ group and affords a Schiff base whose subsequent hydrolysis gives rise to pyridoxamine and L-α-glutamic acid.

Full text of DOI:10.1134/S1070363214070196

ISSN 1070-3632, Russian Journal of General Chemistry, 2014, Vol. 84, No. 7, pp. 1362–1366. © Pleiades Publishing, Ltd., 2014.
Original Russian Text © F.V. Pishchugin, I.T. Tuleberdiev, 2014, published in Zhurnal Obshchei Khimii, 2014, Vol. 84, No. 7, pp. 1167–1171.
Kinetic and Mechanism of Reactions
of L-α-Glutamic Acid and L-Glutamine with Pyridoxal
F. V. Pishchugin and I. T. Tuleberdiev
Institute of Chemistry and Chemical Technology, National Academy of Sciences of Kirgiz Republic,
pr. Chui 267, Bishkek, 720071 Kirgizia
e-mail: pishugin@rambler.ru
Received January 30, 2014
Abstract—The kinetics and mechanisms of condensation of pyridoxal with L-α-glutamic acid and L-glutamine
were studied by UV spectroscopy and polarimetry. L-α-Glutamic acid reacts with pyridoxal to form a Schiff
base whose subsequent hydrolysis gives rise to pyridoxamine and α-ketoglutaric acid. The reaction of L-
glutamine with pyridoxal involves the γ-NH2 group and affords a Schiff base whose subsequent hydrolysis
gives rise to pyridoxamine and L-α-glutamic acid.
Keywords: pyridoxal, amino acids, Schiff bases, glutamine, kinetics
DOI: 10.1134/S1070363214070196
Glutamic acid and glutamine (2,5-diamino-5-
oxopentanoic acid) are actively involved in nitrogen
transfer reactions. The amide nitrogen of glutamine
takes part in many biochemical processes leading to
formation of glucosamine [1], purines, p-amino-
benzoate, and hystidine. Glutamic acid and glutamine
play an important role in the elimination of nitrogen
from organic compounds. The transamination reaction
is the initial step in the catabolism of excess amino
acids.
higher than in the case of L-α-glutamic acid
[рKа (α-NH2) = 9.67; k = 0.0142 min–1], even though
the α-NH2 group in glutamine is less basic. Apparently,
the rate of condensation of pyridoxal with L-α-
glutamic acid is much affected, along with the basicity
of its α-NH2 group at the stages of addition to aldehyde
and dehydration of amino alcohol, by steric factors
(two СОО– groups) which hinder addition to alcohol
and formation of amino alcohol. Evidence for this
sugges-tion is provided by the condensation of L-2-
amino-5-methoxy-5-oxopentanoic acid with pyridoxal
(Fig. 1, curve 3). Methylation of the γ-СООН groups
in glutamic acid accelerates the reaction.
In our previous works [2–7] we described chemical
transformations of a series of amino acids and amines
under the action of pyridoxal and pyridoxal-5'-
phosphate. Kinetic study and isolation of intermediate
and fina reaction products showed that the formation
of Schiff bases and their subsequent transformations
occur in three stages. The rate of each of these stages
depends on the structure of initial, intermediate, and
final products, рН of the medium, solvent,
temperature, and steric and thermodynamic factors.
Glutamine has two nitrogen atoms which, depend-
ing on conditions (рН of the medium, solvent, tempera-
ture) can act as two competing nucleophilic centers.
The γ-NH2 group in glutamine (рKа = 5.65) is less
basic than the α-NH2 group (рKа = 9.13). Therefore, at
first glance, at the stage of amino alcohol formation
pyridoxal should prefer to react by the α-NH2 group of
glutamine. However, at the dehydration stage, the
reverse situation takes place: Water is preferably
eliminated from amino alcohol which is formed by the
reaction of pyridoxal by the γ-NH2 group of the amide
fragment. Since the dehydration stage is a limiting
stage, the contribution of the reaction of pyridoxal by
the γ-NH2 group of glutamine will exceed that of the
Kinetic and mechanistic study of the reactions of
pyridoxal with L-α-glutamic acid and glutamine,
analysis of kinetic curves, and calculation of rate
constants gave unexpected results. The rates of both
reversible and irreversible pyridoxal reactions leading
to Schiff base formation in the case of L-glutamine
[рKа (α-NH2) = 9.13; k = 0.0537 min–1] is ~4 times
1362

KINETIC AND MECHANISM OF REACTIONS
1363
[α]D20°
D
5
15 25 35 45 55
τ, min
τ, min
Fig. 1. Change of the optical densities of mixtures of 0.01 М
solutions of pyridoxal hydrochloride and (1) L-α-glutamic
acid, (2) L-glutamine, and (3) γ-methyl ester of L-α-
glutamic acid with time at the stage of amino acid
formation and dehydration (70% alcoholic aqueous buffer
solutions, рН = 6.5, Т = 20°С).
Fig. 2. Change of the specific rotation angles of 0.04 М
solutions of pyridoxal hydrochloride with (1) L-α-
glutamine, (2) L-α-glutamic acid with time at the stage of
formation and dehydration of (3) amino alcohols, L-α-
glutamic acid and (4) glutamine (70% alcoholic aqueous
buffer solutions, рН = 6.35, T = 20°С).
reaction by the α-NH2 group. To provide evidence for
the suggested routes and schemes of the condensation
of pyridoxal with glutamic acid and its amide, we
performed a kinetic study of these reactions by means
of polarimetry (Fig. 2). In the course of pyridoxal
reaction with L-α-glutamic acid, the negative specific
rotation angles were first decreased and then increased.
Prolonged standing of a mixture of pyridoxal and L-α-
glutamic acid in a solution gave pyridoxamine and α-
ketoglutaric acid. The latter product was isolated and
identified [elemental analysis, 2,4-dinitrophenylhyd-
razone (mp 200°С), negative ninhydrin test]. A dif-
ferent picture was observed in the reaction of pyridoxal
with L-glutamine. At the addition stage, the positive
specific rotation angle rapidly decreased until it
became negative. At the second stage (dehydration),
the negative specific rotation angle increased to a
certain constant value. The fact that the specific
rotation angle changed sign from positive to negative
can be explained by the formation of a new chiral
center at the stage of amino alcohol formation by the
reaction of pyridoxal with the γ-NH2 group of L-
glutamine. The slow increase of the negative specific
rotation angle can be explained in terms of hydrolysis
of Schiff base and formation of pyridoxamine and L-α-
glutamine acid which precipitated and was identified
(elemental analysis, IR spectroscopy, and value of
negative specific rotation).
To find explanation for the resulting data, we have
explored the possible structures of and bond lengths
and atomic charges in the products of the reactions of
pyridoxal with L-α-glutamic acid and L-glutamine by
the α-NH2 groups and with L-glutamine by the γ-NH2
group, obtained by HyperChem [8] with energy and
geometry optimization. It was shown that the reaction
products of pyridoxal with L-α-glutamic acid and L-
glutamine by the α-NH2 group have similar structures
and charges on nodal atoms. The reaction product of
pyridoxal with L-glutamine by the γ-NH2 group has
quite different parameters.
The pyridine fragment in the Schiff bases and
intermediate reaction products (amino alcohols) is
planar. The OH group in the pyridine fragment is
virtually coplanar to the pyridine ring, and the CH2OH
group deviates, in force of its nonlinearity, from the
pyridine ring plane, i.e. this group can serve as a
“probe” for the stereochemistry of the reaction
products. The results of HyperChem calculations
including geometry and energy optimization showed
that the carbonyl group and azomethine fragments in
pyridoxal and Schiff bases formed by condensation by
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 84 No. 7 2014

1364
PISHCHUGIN, TULEBERDIEV
Scheme 1.
OH H COO_
_
COO
*
*
O
H
O
COOH
H
C
N
C
(CH2)2
*
H
CH
(CH2)2
C
C
H
OH
+ NH3
HO
CH2OH
HO
CH2OH
H3C
H3C
+
+
N
N
COO_
*
H
H
H
C
COO _
(+0.011)
COOH
HC
N
C
(CH2)2
COOH
H
N
C
(CH2)2
H
HO
H3C
CH2OH
HO
H3C
CH2OH
H2O
+
NH2
CH2
N
N
H
H
H2O
HO
H3C
CH2OH
+
HOOC
C
(CH2)2 COOH
+
N
O
H
Scheme 2.
COO_
*
_
OH H
*
O
C
COO
O
H
O
*
C
H
C
N
(CH2)2
H
(CH2)2
C
H
C
C
+NH3
NH2
+ NH3
HO
CH2OH
HO
CH2OH
H3C
H3C
+
+
N
N
H
H
+ NH3
COO _
O
*
*
CH
HC
N
C
(CH2)2
C
H
C
N
C
(CH2)2
H
(+0.366)
COO_
OH
CH2OH
+NH3
HO
CH2OH
HO
H2O
H3C
H3C
+
+
N
N
H
H
NH2
HO
O
CH2
C
H2O
(CH2)2
*
HO
H3C
CH2OH
+
_OOC CH
+NH3
+
N
H
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 84 No. 7 2014

KINETIC AND MECHANISM OF REACTIONS
1365
the α-NH2 group are turned by ~90° and reside on the
same side with respect to the “probe,” whereas the ОН
group and amino acid fragment in amino alcohols
reside on different sides from the “probe.”
thermostat with an accuracy of ±0.1°С. Equimolar
amounts of pyridoxal hydrochloride and amino acids
and their amides were dissolved in aqueous alcoholic
buffer solutions and kept for 30 min at a required
temperature. The reaction time was measured starting
from the point of mixing of thermostated solutions of
pyridoxal and amino acids and their amides. Kinetic
measurements were performed in thermostated cells
1.008 mm thick and polarimetric tubes 1.9 dm long.
Reference cells were charged with equimolar solutions
of pyridoxal in the same solvent with the same рН. рН
measurements were performed on an EV-74 ionometer
with an accuracy of ±0.1 units. The condensation rate
constants of pyridoxal with glutamic acid and
glutamine were computed for reversible and
irreversible reactions [9]. The starting and final
products were identified by elemental analysis, UV
and IR spectroscopy, and thin-layer and liquid
chromatography. The IR spectra were measured on a
Nicolet Impact 420 spectrophotometer. The reaction
products were analyzed on a Cole Parmer PLC-20
chromatograph, sorbent С-185μ, eluent H2O–CH3CN
(80 : 20). The structures and atomic charges were
calculated with optimization of geometric and
thermodynamic parameters using HyperChem [8].
In the reaction products of L-glutamine and
pyridoxal by the γ-NH2 group, the ОН group and
amino acid fragments in amino alcohols reside on
different sides with respect to the “probe.” This most
likely explains the differences in the trends in specific
rotation angles (Fig. 2).
The probability of rapid hydrolysis of the Schiff
base by the γ-NH2 group is confirmed by the larger
positive charge on the carbon atom (+0.336) in the
=N–C=O group compared with that on the carbon
atom (+0.011) in the Schiff base by the α-NH2 group.
Chromatography showed that the solutions contain
2 major products of the reaction of pyridoxal with L-
glutamine by the γ-NH2 group: pyridoxamine and
glutamic acid. The reaction products were eluted with
plates and identified by elemental analysis, UV and IR
spectroscopy, and liquid chromatography. The
mechanisms of the reactions of L-α-glutamic acid and
L-glutamine with pyridoxal are shown in Schemes 1
and 2, respectively.
The reaction of pyridoxal with L-α-glutamic acid
was performed by the procedures described in [2–7].
The Schiff base was obtained as yellow crystals
melting with coaling, yield ~83%. The elemental
analysis, UV and IR spectra are consistent with
published data.
Thus, the kinetic study of the reactions of pyridoxal
with L-α-glutamic acid and glutamine, as well as
synthesis and identification of the reaction products
showed that the reactions involves formation of a
Schiff base which undergoes hydrolysis on prolonged
standing of the reaction mixture to form pyridoxamine
and α-ketoglutaric acid.
Reaction of pyridoxal with glutamine. To a
mixture of 0.0103 g of pyridoxal hydrochloride and
0.0073 g of glutamine, 8 mL of 96% ethanol was
added. The mixture was heated at 50°С until the
reagents dissolved completely. The reaction progress
was monitored by UV spectroscopy (λmax 350 and
420 nm) and TLC. After reaction completion the
solvent was let to evaporate at room temperature.
Yield 0.082 g (~52%), hygroscopic dark red substance,
mp >310°С. IR spectrum (KBr), ν, cm–1: 3187 (NH),
1665 and 1540 (amide I, amide II). The UV maxima
are stronger and shifted blue by 7–10 nm compared to
the spectrum of the condensation product of pyridoxal
with L-α-glutamic acid. Found, %: С 47.4; Н 5.2; N
12.6. C13H17N3O5·HCl. Calculated, %: С 47.2; H 5.1;
N 12.7.
The reaction of glutamine with pyridoxal occurs
predominantly by the γ-NH2 group of the amide
fragment to form a Schiff base; the latter rapidly
rearranges into a quinoid structure whose hydrolysis
forms pyridoxamine and L-α-glutamic acid.
EXPERIMENTAL
Pyridoxal hydrochloride of chemical grade obtained
from Ferak Berlin and amino acids and their amides
obtained from Reanal were used. Buffer solutions were
prepared by conventional procedures. Kinetic measure-
ments were performed on a Spektonom-204 spectro-
photometer and a Digi Pol DS Automatic Sachari-
meter. Reaction mixtures were thermostated in a UH-8
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PISHCHUGIN, TULEBERDIEV
5. Pishchugin, F.V. and Tuleberdiev, I.T., Russ. J. Gen.
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