Kinetics and mechanism of transaldimination of amino acids and aromatic amines with pyridoxal

Article abstract of DOI:10.1134/S1070363213060121

Kinetics and mechanism of transaldimination of amino acids and aromatic amines with pyridoxal have been studied by means of UV spectroscopy and polarimetry. It has been shown that aminal intermediates are formed in reaction of the Schiff's bases with p-am

Full text of DOI:10.1134/S1070363213060121

ISSN 1070-3632, Russian Journal of General Chemistry, 2013, Vol. 83, No. 6, pp. 1077–1080. © Pleiades Publishing, Ltd., 2013.
Original Russian Text © F.V. Pishchugin, I.T. Tuleberdiev, 2013, published in Zhurnal Obshchei Khimii, 2013, Vol. 83, No. 6, pp. 946–949.
Kinetics and Mechanism of Transaldimination
of Amino Acids and Aromatic Amines with Pyridoxal
F. V. Pishchugin and I. T. Tuleberdiev
Institute of Chemistry and Chemical Technology, Kyrgyz Republic National Academy of Sciences,
pr. Chui 267, Bishkek, 720071 Kyrgyzstan
e-mail: pishugin@rambler.ru
Received May 28, 2012
Abstract―Kinetics and mechanism of transaldimination of amino acids and aromatic amines with pyridoxal
have been studied by means of UV spectroscopy and polarimetry. It has been shown that aminal intermediates
are formed in reaction of the Schiff’s bases with p-aminobenzoic acid and β-alanine. The structure of aminal
and Schiff’s base is determined by the spatial arrangement of the amino acid and aromatic fragments with
respect to the pyridine ring plane. The presence of ОН and СН2–ОН groups in the o-positions in pyridoxal
structure turns amino groups by 90° with respect to the pyridine ring. The scheme of Schiff’s bases
transaldimination by amino acids and biological amines has been developed according to stereospecific,
energy, and geometric factors.
DOI: 10.1134/S1070363213060121
In our previous studies [1–4] it was demonstrated
that the reaction of pyridoxal with amino acids
proceeded through 3 kinetically different stages:
(1) NH2 group addition to the pyridoxal carbonyl
group with the formation of the amino alcohol (very
fast stage); (2) amino alcohol dehydration with the
formation of the Schiff’s base (slow stage); (3) Schiff’s
base rearrangement into a quinoid structure by α-
hydrogen elimination, with subsequent hydrolysis
giving pyridoxamine and keto acids.
fast and are not always unambiguous. Due to this, the
biochemical processes mechanisms are often proved
by studies of interaction between model enzyme and
substrate fragments under the controlled conditions.
In order to fill in the gap in our knowledge on
transaldimination mechanism, in this work we studied
kinetics and mechanism of the Schiff’s bases interac-
tion with amino acids and aromatic amines under
varied conditions. To do so, we chose the following
objects: p-aminobenzoic acid, β-alanine, and the cor-
responding Schiff’s bases, products of p-aminobenzoic
acid and β-alanine condensation with pyridoxal. There
are no chiral centers in these compounds, and their
optical absorbance bands do not overlap [λmax (pyrid-
oxalidene–p-aminobenzoic acid) = 370 nm, and
λmax (pyridoxalidene-β-alanine) = 340 and 430 nm].
It is known that new Schiff’s bases formation is
likely to proceed via amino group addition to the C=N+
group (via transaldimination reaction), but not via addition
to the C=O group of PLP-dependent enzymes [5, 6].
The stage of addition to the L-lysine condensation
product is followed by lysine ε-amino group elimina-
tion. The indirect proof of this mechanism is the
assumption suggested in [5] that the addition to the
HC=NH+ bond proceeds faster than the addition to the
HC=O group. According to [5], this agrees with the
disappearance of the positive circular dichroism upon
addition of the substrate to the coenzyme. After having
the substrate reacted, the circular dichroism reappeared.
Kinetics and mechanism of pyridoxylidene-β-alanine
interaction with p-aminobenzoic acid were studied
following the absorbance at λmax = 430 nm. In the
initial stage the absorbance decreased sharply, this was
followed by the second stage accompanied by slow
continuous decrease in the absorbance. On the contrary,
during the reaction of pyridoxalidene-p-aminobenzoic
acid with β-alanine, the absorbance at λmax = 430 nm
initially sharply increased and then continued to grow
slowly (Figs. 1, 2).
However, the discussed mechanism has been still
an open question. Seemingly, it can be hardly solved
by studies of the enzyme reactions, as they proceed
1077

1078
PISHCHUGIN, TULEBERDIEV
D
D
D
τ, min
Fig. 1. Kinetics of the absorbance (λmax = 430 nm) change
upon interaction of pyridoxaliden-β-alanine with p-
aminobenzoic acid (1) and that of pyridoxaliden-p-
aminobenzoic acid with β-alanine (2), in aqueous ethanol
(90 vol % of EtOH) buffer solution (рН 7.0, 20°С). (1)
addition stage, (2) the stage of amino acid or p-
aminobenzoic acid elimination.
λ, nm
Fig. 2. Optical spectra of the reaction mixture of pyrid-
oxaliden-β-alanine and p-aminobenzoic acid; in aqueous
ethanol (90 vol % of EtOH) buffer solution (рН 6.9, 20°С).
Numbers near the corresponding curves indicate the
reaction time in min.
Analysis of the kinetic curves led to assumption
that in the first stage (sharp increase or decrease of the
absorbance) the addition of the NH2 group of amino
acid or p-aminobenzoic acid to the Schiff’s base
occurred resulting in the formation of an aminal
intermediate. In the second stage the amino acid or p-
aminobenzoic acid was eliminated to give the final
product: pyridoxalidene-p-aminobenzoic or pyridoxal-
idene-β-alanine, respectively. The suggested trans-
aldimination mechanism was proved by studies of the
pyridoxalidene-β-alanine reaction with m-aminobenzoic
acid. The final product of this reaction, pyridoxalidene-
m-benzoic acid, was poorly soluble and thus
precipitated from the reaction mixture. This final pro-
duct precipitate was separated and characterized by
elemental analysis and IR spectroscopy.
At this research point, a question appeared: having
known from literature and our data that the
intermediate (aminal) formed at the first stage was the
same in both studied reactions, why during the
pyridoxalidene-β-alanine interaction with p-amino-
benzoic acid the reaction mixture absorbance initially
decreased, while the reaction of pyridoxalidene-p-
benzoic acid with β-alanine was accompanied by the
absorbance increase. To clear this out, kinetics and
mechanism of transaldimination were additionally
studied by polarimetry. At the stage of β-alanine or
pyridoxalidene-p-benzoic acid addition to the Schiff’s
base, a chiral center should have appeared. Data in Fig. 3
show that in the initial stage of pyridoxalidene-p-benzoic
acid interaction with β-alanine, a product with positive
specific angle of optical rotation was rapidly formed,
and then in the course of the reaction (p-aminobenzoic
acid elimination) the positive optical rotation angle
steadily decreased. On the contrary, in the reaction of
pyridoxalidene-β-alanine with p-aminobenzoic acid, a
product with negative optical rotation angle was
initially formed, with the absolute value of the rotation
angle steadily decreasing as β-alanine was eliminated.
[α]D20°
τ, min
Fig. 3. Time dependence of the change in the specific
optical rotation angle at the interaction of pyridoxaliden-p-
aminobenzoic acid with β-alanine (1) and that of pyrid-
oxaliden-β-alanine with p-aminobenzoic acid (2), in
aqueous ethanol (90 vol % of EtOH) buffer solution (рН
6.7, 20°С).
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KINETICS AND MECHANISM OF TRANSALDIMINATION
1079
The analysis of kinetic data from UV spectroscopy
and polarimetry measurements, as well as structural
analysis of the intermediates (amino alcohols) and final
product revealed that in the Schiff’s bases of pyrid-
oxalidene-p-aminobenzoic acid and pyridoxalidene-β-ala-
nine, the aminal group was turned by 90° with respect
to the pyridine ring plane. Due to sterical hindrance (pres-
ence of the OH and CH2OH groups in o-positions), the
addition of amino acid or aromatic amine to the
Schiff’s base was stereospecific along the C=NH group
plane. Thus, the addition was accompanied by very
fast change of the solutions absorbance (either sharp
increase or sharp decrease) and appearance of the
chiral centers. Then, seemingly, rotational isomer was
formed due to optimization of geometric and energy
parameters accompanying the elimination stage (according
to HyperChem software data). The transaldimination
mechanism may be presented as follows.
R
N
R
R'
N+H2
R'
H
N
H
HN
H
_O
C
H
O
C
C
_O
R'NH2
CH2OH
CH2OH
CH2OH
_RNH2
H3C
H3C
H3C
+
+
+
N
N
N
H
H
H
R
R
R'
R'
H2N+
HN
H
N
H
N
+
H
C
C
H
C
_O
_O
_O
RNH2
CH2OH
CH2OH
CH2OH
_R'NH2
H3C
H3C
H3C
+
+
+
N
N
N
H
H
H
R = 4-C6H5COOH; R' = CH2CH2COOH.
Why in reactions of pyridoxalidene-p-aminobenzoic
acid with β-alanine and of pyridoxalidene-β-alanine
with p-aminobenzoic acids the aminals with the
opposite signs of the optical rotation angle and
different kinetic profiles of their absolute values were
formed? We did not succeed in the aminals isolation,
even at low reaction temperature, as the intermediates
were highly unstable, and their structural studies were
thus impossible. To clear up this question, the
structures of aminals and Schiff’s bases were studied
in the HyperChem software, after the optimization of
their geometric and energy parameters. As it was
indicated earlier, C=N+HR and RH2N+–C–NHR'
groups were turned by ~90° with respect to the
pyridine ring plane, and it was convenient to orient the
structures so that the pairs of carbon atoms in o- and
m- positions of the pyridine ring were superimposed.
According to the structural data, o-OH group was
located approximately in the plane of the pyridine ring
(conventionally “left side”), whereas the o-CH2OH
group, being non-linear, was out of the pyridine ring
plane (conventionally “right side”).
The aromatic fragment was located to the left in the
aminals and the Schiff’s bases, whereas the amino acid
fragment was located to the right. Thus, upon the
interaction of pyridoxalidene-p-aminobenzoic acid with
β-alanine and that of pyridoxalidene-β-alanine with p-
aminobenzoic acid, the aminal intermediates with dif-
ferent location of the aromatic and amino acid frag-
ments were formed: R(D+) and S(L–). According to the
Kahn-Prelog rule, this may be represented as follows.
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 83 No. 6 2013

1080
PISHCHUGIN, TULEBERDIEV
R1 (88)
R2 (136)
R1 (88)
R2 (136)
R3 (167)
R3 (167)
S (L-)
R1 – amino acid fragment, R2 – p-aminobenzoic acid fragment, R3 – pyridine fragment.
R (D+)
Subsequently, the elimination of one of the
hydrochloride (0.103 g) and p-aminobenzoic acid
(0.0685 g). The resulting mixture was heated up to 50°С
during 20 min (till complete dissolution) using the
water temperature bath. Reaction course was
monitored with the UV spectrophotometer and by thin-
layer chromatography. The reaction was run till the
constant absorbance at λmax = 370 nm was reached, and
till the TLC spots corresponding to initial reactants
disappeared. Intensive orange coloring appeared upon
mixing the reactants solutions. The mixture was
evaporated at room temperature till the red precipitate
was formed. Product yield 0.144 g (88%), mp 280°С
(decomp.). UV spectrum: λmax = 370 nm. IR spectrum
(KBr), ν, cm–1: 1620 (C=N), 1385, 1512 (С=О, СОО–).
Found, %: С 55.1; Н 5.05; N 8.1. C15H14N2O4·HCl.
Calculated, %: С 55.9; Н 4.9; N 8.6.
fragments from the aminal structure occurred,
accompanied with the formation of rotational isomer,
the final product with optimal energy and geometric
parameters. These experimental data seem to explain
the relation between the optical rotation angle and its
sign with the structures of intermediates and final
products as well as with UV spectroscopy data.
EXPERIMENTAL
Pyridoxal hydrochloride of the chemically pure
grade (Ferak Berlin) and amino acids (Reanal,
Hungary, England) were used. Buffer solutions were
prepared according to published procedures. Reaction
kinetics were followed by measurements using
SpectroMOM-204 spectrophotometer and DigiPol DS
saccharimeter (USA). Temperature control of the
reaction mixtures in the spectrophotometer cells and
polarimeter tubes was assured with UH-8 temperature
bath, temperature accuracy being of ±0.1°C. In kinetic
studies, equimolar amounts of the reactants were
dissolved separately in aqueous ethanol buffer
solutions and incubated for 30 minutes at reaction
temperature. The moment of the incubated reactants
mixing was taken as the reaction start time.
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3. Pishchugin, F.V. and Tuleberdiev, I.T., Russ. J. Gen.
Chem., 2009, vol. 79, no. 1, pp. 117–120.
Condensation and transaldimination products were
prepared according to previously published procedures
[1–4]. Initial and final products were characterized by
elemental analysis, UV and IR spectroscopy.
4. Pishchugin, F.V. and Tuleberdiev, I.T., Russ. J. Gen.
Chem., 2010, vol. 80, no. 9, pp. 1836–1840.
5. Metzler, D.E., Biochemistry: The Chemical Reactions of
Living Cells, New York: Academic Press, 1977.
Pyridoxalidene-p-aminobenzoic acid. Ethanol,
6. Braunshtein, A.E. and Shemiakin, M.M., Biokhim.,
1953, vol. 18, no. 4, pp. 393–411.
6 ml, (96 vol %) was added to a mixture of pyridoxal
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