Kinetics and mechanism of the condensation of pyridoxal hydrochloride with L-tryptophan and D-tryptophan, and the chemical transformation of their products

Article abstract of DOI:10.1134/S0036024417100302

The kinetics and mechanism of interaction between pyridoxal and L-tryptophan, D-tryptophan, and their derivatives are studied. It is found that condensation reactions proceed via three kinetically distinguishable stages: (1) the rapid intraplanar addition of the NH₂ groups of the amino acids to pyridoxal with the formation of amino alcohols; (2) the rotational isomerism of amino alcohol fragments with their subsequent dehydration and the formation of a Schiff base with a specific configuration; (3) the abstraction of α-hydrogen in the product of condensation of pyridoxal with L-tryptophan, or the abstraction of СО₂ in the product of condensation of pyridoxal with D-tryptophan with the formation of quinoid structures, hydrolysis of which results in the preparation of pyridoxamine and keto acid or pyridoxal and tryptamine, respectively. Schiff bases resistant to further chemical transformations are formed in the reaction with tryptophan methyl ester.

Full text of DOI:10.1134/S0036024417100302

ISSN 0036-0244, Russian Journal of Physical Chemistry A, 2017, Vol. 91, No. 10, pp. 1851–1854. © Pleiades Publishing, Ltd., 2017.
Original Russian Text © F.V. Pishchugin, I.T. Tuleberdiev, 2017, published in Zhurnal Fizicheskoi Khimii, 2017, Vol. 91, No. 10, pp. 1648–1652.
CHEMICAL KINETICS
AND CATALYSIS
Kinetics and Mechanism of the Condensation of Pyridoxal
Hydrochloride with L-Tryptophan and D-Tryptophan,
and the Chemical Transformation of Their Products
F. V. Pishchugin* and I. T. Tuleberdiev
Institute of Chemistry and Chemical Technology, National Academy of Sciences of the Kyrgyz Republic,
Bishkek, 720071 Kyrgyz Republic
*
e-mail: pishugin@rambler.ru
Received November 11, 2016
Abstract—The kinetics and mechanism of interaction between pyridoxal and L-tryptophan, D-tryptophan,
and their derivatives are studied. It is found that condensation reactions proceed via three kinetically distin-
guishable stages: (1) the rapid intraplanar addition of the NH groups of the amino acids to pyridoxal with the
2
formation of amino alcohols; (2) the rotational isomerism of amino alcohol fragments with their subsequent
dehydration and the formation of a Schiff base with a specific configuration; (3) the abstraction of α-hydro-
gen in the product of condensation of pyridoxal with L-tryptophan, or the abstraction of СО in the product
2
of condensation of pyridoxal with D-tryptophan with the formation of quinoid structures, hydrolysis of
which results in the preparation of pyridoxamine and keto acid or pyridoxal and tryptamine, respectively.
Schiff bases resistant to further chemical transformations are formed in the reaction with tryptophan methyl
ester.
Keywords: kinetics, mechanism, pyridoxal hydrochloride, L- and D-tryptophan, UV spectrometry and pola-
rimetry
DOI: 10.1134/S0036024417100302
INTRODUCTION
the kinetics of chemical processes and isolating their
intermediate and final products that the formation of
Schiff bases and their chemical transformations pro-
ceed in three stages. The first stage is the addition of
the amino group of an amino acid to the carbonyl
group of pyridoxal to form the intermediate product,
an optically active amino alcohol (a sharp drop in
absorbance). This stage proceeds very rapidly, and the
rates of interaction between the two tryptophan iso-
mers are apparently close. The second stage is the
dehydration of the amino alcohol to form a Schiff base
The stereochemistry of amino acids plays a key role
in the structure of human and animal proteins, pep-
tides, and enzymes. L-amino acids are components of
these biological objects. The stereospecificity of the
between enzymes and substrates of specific configura-
tions is associated with this. D-Amino acids are pres-
ent in many peptides produced by microorganisms
and form part of the biopolymers of their cell tissues.
The inclusion of D-α-amino acids in the structures of
human proteins and enzymes has a fatal outcome.
Tryptophan forms part of the polypeptides of plant
and animal organisms. The hydroxylation of trypto-
phan with subsequent decarboxylation results in the
formation of serotonin, which plays an important role
as a brain neurotransmitter; the failure of its normal
metabolism in the body results in schizophrenia. The
decarboxylation of tryptophan leads to the formation
of tryptamine, an intermediate product in the forma-
tion of lysergic acid [1].
(a slow drop in absorbance); and the third stage is the
elimination of α-hydrogen or СО , followed by their
2
hydrolysis to form the final products (the slowest
stage). The rates of these limiting states depend on fac-
tors of stereochemical and energy. It is of definite
interest to investigate the kinetics and mechanism of
interaction between pyridoxal and L-tryptophan, D-
tryptophan, and their derivatives (Fig. 1), depending
on the structure of the initial, intermediate, and final
products.
Our earlier works found that chemical transforma-
tions of amino acids and bioamines under the action of
pyridoxal and pyridoxal-5'-phosphate [2–7] depend
on their structure, the рН of the medium, the tem-
EXPERIMENTAL
Chemically pure pyridoxal hydrochloride (Ferak
perature, and the solvent. It was found by measuring Berlin), amino acids, and amides of amino acids
1851

1852
PISHCHUGIN, TULEBERDIEV
D
Synthesis of Tryptamine Hydrochloride
4
3
1.4
1.0
0.6
0.2
Mixtures of equimolar 0.01 М pyridoxal hydro-
chloride and D-tryptophan in a 90% buffer solution
were kept for 18 h at Т = 15°C. In preparing the solu-
tions, the mixtures turned an intense yellow and
changed their color to orange over time. The color
then became less intense, and a white precipitate
formed. The precipitate was filtered off, washed with
alcohol, and dried to a constant weight. The yield was
2
1
0.0088 g (23.3%); Т > 210°C (the precipitate black-
m
ened as it decomposed). The product yielded a quali-
−
10
30
50
70
90 τ, min
tative reaction for Cl with a solution of AgNO and a
3
qualitative reaction with Br (violet). The final prod-
2
Fig. 1. Kinetics of the interactions between mixtures of
.01 М solutions of pyridoxal hydrochloride with (1) L-
uct was poorly soluble in alcohol (IR spectrum (KBr),
0
−1
ν, cm : 3150–3350 (NH ), 2250–2700 (>NH), 1625,
2
tryptophan methyl ester λ
355 nm, (2) tryptamine, (3)
max
1
580 (C=C); UV spectrum, λ : 280 nm).
L-tryptophan, and (4) D-tryptophan at the stage of amino
alcohol dehydration (430 nm, 90% aqueous–alcoholic
buffer solution, рН 7.2, 15°C).
max
Found, %:
Calculated, %: С 61.2,
С 61.1,
Н 6.1,
Н 6.2,
N 14.2, Cl 18.2
N 14.2, Cl 18.4
(
Reanal, Great Britain) were used in this work. Buffer
solutions were prepared according to the standard pro-
cedure. The reaction kinetics was measured on a
Spektonom-204 spectrophotometer and a Digi Pol
RESULTS AND DISCUSSION
The data in Fig. 1 show that at the stage of amino
DS automatic saccharimeter. The reaction mixtures alcohol dehydration, D-tryptophan is more active
were thermostated using a UH-8 thermostat with an than L-tryptophan. This is explained by the great
accuracy of ±0.1°C. Weighed amounts of pyridoxal influence of the structures of amino acids, their initial
and final products, and their kinetic and thermody-
namic factors. With interaction between pyridoxal and
hydrochloride and amino acids and their methyl esters
were dissolved in equimolar quantities in aqueous–
alcoholic buffer solutions and kept for 30 min at a
specified temperature. The moment of mixing the
thermostatted solutions of pyridoxal, amino acids,
and methyl esters of amino acids was used to mark the
start of the reaction. Kinetic measurements were made
in thermostatted cells 1.008 mm thick and polarimet-
ric tubes 1.9 dm long. Since the UV spectra of solu-
tions of pyridoxal vary depending on the рН of the
medium and the solvent, equimolar solutions of pyri-
doxal in the same solvent with the same value of the
рН of the medium were placed into reference cells.
The рН of the solutions was measured on an EV-74
ionometer with an accuracy of ±0.1 рН unit. The rate
constantsofthecondensationofpyridoxalwithL-tryp-
tophan, D-tryptophan, and their esters were calcu-
lated on a computer for reversible and irreversible
reactions [8]. The initial and final products were iden-
tified via elemental analysis, UV and IR spectroscopy,
and TLC. The IR spectra were recorded on a Nicolet
Impact 420 IR spectrophotometer. The products of
interaction were analyzed on a PLC-20 liquid chro-
matograph (Cole Parmer) with С185 μm sorbent; the
–
L-tryptophan, the α-hydrogen or СОО of the amino
acid fragment in a Schiff base should, according to the
Danatan convention [1], be in a position favorable for
their abstraction and the Schiff base’s transition to a
quinoid structure, the subsequent hydrolysis of which
results in the formation of the final products. Upon
interaction between pyridoxal and D-tryptophan, a
Schiff base is formed in which the amino acid frag-
ment is turned by 90° relative to the plane of the pyri-
dine ring; as a result, a Schiff base with preferable
abstraction of СО is formed with the subsequent for-
2
mation of a quinoid structure, hydrolysis of which
results in the preparation of pyridoxal and tryptamine.
The polarimetric data on the kinetics of the condensa-
tion of pyridoxal with L-tryptophan and D-trypto-
phan serve as proof of the proposed postulate (Fig. 2).
The results from the above measurements show
that at the first stage of condensation, there is a rapid
increase or decrease in the observed angles of rotation
of a mixture of solutions, and these angles gradually
change to zero over time. At the stage of amino alcohol
formation, new chiral centers with angles of rotation
different in magnitude and sign form in both cases
(a fast stage). If we start from the premise that nucleo-
philic reagents attack the plane of the carbonyl group
from both sides with equal probability, there should be
eluent was H O : CH CN = 80 : 20%. The structure
2
3
and values of charges on the atoms were determined by
optimizing the geometrical and thermodynamic fac-
tors using the Hyper Chem program. The Schiff bases no change in the magnitudes and signs of the angles of
were synthesized according to the procedures rotation, since racemates are in this case formed.
described in [2–7].
These contradictions can only be explained by assum-
RUSSIAN JOURNAL OF PHYSICAL CHEMISTRY A
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KINETICS AND MECHANISM OF THE CONDENSATION
1853
amino alcohols caused by the optimization of energy
and geometrical parameters results in their dehydra-
tion with the formation of specific structures of Schiff
bases that facilitate the possible abstraction of the α-
α
2
1
hydrogen or СО groups. At the third stage, the Schiff
2
bases are rearranged with the formation of quinoid
structures, the hydrolysis of which results in the for-
mation of the final products. Data on the structures of
amino alcohols and Schiff bases, obtained using the
Hyper Chem program with allowance for the optimi-
zation of geometrical and energy parameters, confirm
this assumption. The results from these studies showed
that the products of the condensation of pyridoxal
1
0
4
5
3
−
1
2
3
(
amino alcohols and Schiff bases) with L-tryptophan
and D-tryptophan differed from each other in the
positions of the amino acid fragments relative to the
plane of the pyridine ring of pyridoxal. To prove the
proposed scheme of the mechanism, the final prod-
ucts were synthesized and identified via elemental
analysis, qualitative reactions, UV and IR spectros-
copy, and TLC.
2
−
−
0
2
4
6
12
16
20
24 τ, h
Fig. 2. Kinetics of the change in the observed angles of
rotation of mixtures of 0.04М solutions of pyridoxal
hydrochloride with (1) D-tryptophan, (2) L-tryptophan,
It was noted that when mixtures of solutions of pyr-
idoxal hydrochloride with L-tryptophan and D-tryp-
tophan were kept, they turned an intense yellow with
(
3) methyl ester, (4) D-tryptophan, and (5) L-tryptophan
over time (70% aqueous–alcoholic buffer solution;
λ
of 350 and 430 nm. When the mixtures were kept
max
рН 6.1; 20°C).
for long periods, sediments precipitated from them
that were isolated and identified via elemental analysis
ing that the nucleophilic attack on the carbonyl group and UV and IR spectroscopy. The procedure for syn-
of pyridoxal occurs along its plane to form an interme- thesizing and identifying the precipitate that is the
diate product, optically active amino alcohol (the first product of interaction between pyridoxal and L-tryp-
tophan was described in [2]. The precipitate that is the
product of interaction between D-tryptophan and
pyridoxal (tryptamine hydrochloride) was isolated,
purified, and analyzed via elemental analysis and UV
and IR spectroscopy. The schemes of the interaction
stage is a rapid change in the absorbance of the mix-
ture of solutions (Fig. 1) or their angles of rotation
(
Fig. 2)) with two chiral centers of a specific structure
in close vicinity.
The second (slow) stage then proceeds. The rota- between pyridoxal and L-(1) and D-(2) tryptophans
tional isomerism of the optically active structures of can be presented as
−
COO
R C H
H
*
*
−
R C COO
N
+H N CH
O
H
L-Trp
NH2
*
OH
*
+
C
H H C
+
R
C
COOH
HO
H C
CH OH
H
−O
CH OH −H2O
−O
CH OH
2
2
2
H C
3
H C
3
3
+NH
+NH
+NH
R C COO−
N
+
NH
3
H
C
CH2
−O
H C
3
CH OH H O
−O
H C
CH OH
2
2
2
+
R C COOH
O
3
+NH
NH
Scheme 1.
RUSSIAN JOURNAL OF PHYSICAL CHEMISTRY A Vol. 91 No. 10 2017

1854
PISHCHUGIN, TULEBERDIEV
−
COO−
COO
*
R CH
*
R C H
H
N
N
H
O
H
D-Trp
H
OH
C
HC
*
H
O
C
*
+
R
C
COOH
HO
H C
CH OH
NH2
HO
H C
3
CH OH −H2O
CH OH
2
2
2
H C
3
3
+NH
+NH
CH R
+NH
CHR
2
N
N
H
H
H
O
H
O
C
H
O
C
C
CH OH (+H+)
(+H O) HO
CH OH
2
−
CO2
2
2
+
R CH NH
2 2
H C
3
H C
3
H C
3
+NH
Scheme 2.
+NH
NH
Studying the kinetics and mechanism of the inter-
The products of the condensation of L-tryptophan
action between L-tryptophan methyl ester and pyri- methyl ester with pyridoxal were somewhat unexpect-
doxal is of definite interest. In earlier works, we edly resistant to further chemical transformation. The
showed that the higher the basicity of the NH group results from our study and analysis of the kinetic mea-
2
surements and structure of the final products showed
that a Schiff base resistant to chemical transformations
formed during the condensation of pyridoxal with
tryptophan methyl ester.
of the amino acid, the higher the rate at which the
amino acid is added. An inverse dependence is
observed at the stage of amino alcohol dehydration;
i.e., the higher the basicity of the NH group, the lower
2
the rate of dehydration. The data presented in Fig. 1
show that the rate of amino alcohol dehydration in the
products of interaction between pyridoxal and L-tryp-
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−1
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2
2
001), Vol. 2.
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1
of its methyl ester κ = 0.0662 min . Tryptamine falls
in the middle, in the activity series κ = 0.0837 min .
2
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−1
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2
3
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The higher reactivity of the condensation of pyri-
doxal with L-tryptophan when compared to its methyl
ester at the stage of amino alcohol dehydration is
apparently explained by the higher accepting ability of
the СООН group, relative to the СООСН group
3
during the abstraction of water from the amino alco-
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explained by the lahigherrger value of рK of the NH
а
2
group of the former when compared to рK of the lat-
1963).
а
ter. Tryptamine with pyridoxal forms a Schiff base that
is resistant to further chemical transformation.
Translated by E. V. Boltukhina
RUSSIAN JOURNAL OF PHYSICAL CHEMISTRY A
Vol. 91
No. 10
2017