Kinetics studies on thermal isomerization of β-N-oxalyl-L-α,β- diaminopropionic acid by capillary zone electrophoresis

Article abstract of DOI:10.1039/a902229e

The reciprocal thermal isomerization in aqueous solution between neurotoxic nonprotein amino acid β-N-oxalyl-L-α,β-diaminopropionic acid (β-ODAP) in seeds of Lathyrus sativus (LS; grass pea) and its nontoxic isomer α-N-oxalyl-L-α,β-diaminopropionic acid (α-ODAP) was studied using capillary zone electrophoresis (CZE) in the range from pH = 2.5 to pH = 13 at T = 75 °C, and a decrease of the equilibrium concentration ratio of α-ODAP with the increase of pH of the solution was found. In the range of temperatures 55-95 °C. The rate constants have been measured and thermodynamic parameters such as activation energy (E(a)) and the pre- exponential factor (A) have been calculated.

Full text of DOI:10.1039/a902229e

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Kinetics studies on thermal isomerization of
b-N-oxalyl-L-a,b-diaminopropionic acid by capillary zone
electrophoresis
Liang Zhao,a Zhixiao Li,b Guanbin Li,a Xingguo Chena and Zhide Hu*a
a Department of Chemistry, L anzhou University, L anzhou 730000, China
b National L aboratory of Applied Organic Chemistry, L anzhou University, L anzhou 730000,
China
Received 22nd March 1999, Accepted 14th June 1999
The reciprocal thermal isomerization in aqueous solution between neurotoxic nonprotein amino acid b-N-
oxalyl-L-a,b-diaminopropionic acid (b-ODAP) in seeds of L athyrus sativus (LS; grass pea) and its nontoxic
isomer a-N-oxalyl-L-a,b-diaminopropionic acid (a-ODAP) was studied using capillary zone electrophoresis
(CZE) in the range from pH \ 2.5 to pH \ 13 at T \ 75 ¡C, and a decrease of the equilibrium concentration
ratio of a-ODAP with the increase of pH of the solution was found. In the range of temperatures 55È95 ¡C,
The rate constants have been measured and thermodynamic parameters such as activation energy (E ) and the
a
pre-exponential factor (A) have been calculated.
the thermal isomerization of b-ODAP at di†erent tem-
1
Introduction
peratures and pHs. The activation energy (E ) and pre-
a
exponential factor (A) of the reaction was computed. The low
The seeds of L athyrus sativus (LS; grass pea) provide a highly
desirable source of protein in certain impoverished regions of
Asia and Africa.1,2 However, excess consumption of the seeds
of LS can cause human neurological disorder, which is
believed to be caused by non-protein amino acid b-N-oxalyl-
L-a,b-diaminopropionic acid (b-ODAP).3 Various processing
and cooking methods have been employed to bring down the
toxicity of seeds of LS.4 Thermal isomerization was used as
one of the detoxiÐcation methods by transforming (b-ODAP)
to its innocuous isomer a-N-oxalyl-L-a,b-diaminopropionic
acid (a-ODAP) in the aqueous solution of b-ODAP.
efficiency of thermal isomerization detoxiÐcation could be
partly explained by experimental results.
Experimental
Instruments
CZE was carried out using a Waters Quanta 4000 system
(Waters Chromatography Division of Milford, MA, USA).
The temperature is 22 ^ 1 ¡C. Separation of a- and b-ODAP
was performed in uncoated fused silica capillaries manufac-
tured by Waters Accasep. Capillaries of 75 lm id, 47.4 cm
e†ective length (55 cm total length) were used. Data acquisi-
tion was carried out with a Maxima820 chromatography
workstation.
b-ODAP H a-ODAP
(1)
The equilibrium concentration between a- and b-ODAP has
been reported to be in the ratio of 35 : 65 at 55 ¡C,5 30 : 70 at
room temperature and 40 : 60 at 55È60 ¡C, and the transform-
ation reaction from b-ODAP to a-ODAP in solution of
b-ODAP (pH \ 2.3) was quicker than in solution of sodium
salt of b-ODAP (pH 6.6).6 It is worth noting that these ratios
were obtained in the aqueous solution of b-ODAP at the pH
of 2.3. However, experimental results reported herein show
that the equilibrium concentration ratio between a- and
b-ODAP decreased with the increase of pH of solution. Since
food made of seeds of LS has much lower acidity than that of
aqueous solution of b-ODAP, it is necessary to investigate the
thermal isomerization of b-ODAP at di†erent acidities in
detail.
Capillary zone electrophoresis (CZE) is a convenient tool
for determination of some physical chemistry constants, and
this technology has been employed for the determination of
dissociation constant (pK )7,8 and kinetics of refolding and
aggregation of proteins.9 As far as we know, there is no
kinetic study using CZE on isomerization or racemization
reported. Because CZE is sensitive and fast to determine the
concentration ratio of a- and b-ODAP,10 this method was
employed here to measure the rate constants (k and k ) for
Separation and quantitation procedure
Separation was performed with 0.010 M borate bu†er,
pH \ 9.2. An applied voltage of 21 kV was employed, and
detection wavelength was set at 185 nm. Samples were intro-
duced from the anodic end of the capillary by hydrostatic
injection where the sample vial was raised by 10.0 cm for 5 s.
The migration time for a- and b-ODAP were 6.28 min and
6.70 min, respectively. A 1 min wash cycle with 0.5 M NaOH
solution followed by 1 min distilled water, and 1 min separa-
tion bu†er was necessary to condition the capillary.
The concentration ratio of a- and b-ODAP was calculated
by
C /C \ kS /S
(2)
where C /C is concentration ratio of a- and b-ODAP, S /S is
a
b
a b
a
a
b
a b
peak area ratio of a- and b-ODAP, k is a constant. To
measure the value of k, NMR spectroscopy6,11 was employed
to determine C /C using a mixture of a and b-ODAP, and
a
b
CZE was used to obtain the value of S /S of this sample.
1
~1
a b
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3771

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Then k was calculated by eqn. (2). In this work the constant k
is 1.12.
k1
b-ODAP A8B a-ODAP
k~1
t \ t
t \ t
a [ x
a [ x
x
Reagent
x
e
e
e
b-ODAP was extracted from LS as in ref. 12. Bu†ers of
pH \ 13.0 were prepared by diluting 1 M NaOH, bu†ers of
pH \ 2.5, pH \ 5.0, pH \ 7.0 were 0.05 M phosphate, bu†ers
of pH \ 9.0, pH \ 11.0 were 0.05 M borate, the required pH
or bu†er was adjusted by H PO and NaOH. Unless other-
Subscript e indicates equilibrium, 1 indicates reaction from
b-ODAP to a-ODAP and [1 indicates reaction from
a-ODAP to b-ODAP. The rate constant (k and k ) was
1
~1
calculated by using the equations of Manfred,14
3
4
wise speciÐed, all chemicals were of analytical reagent grade.
lnM(x [ x )/(x [ x )N \ (k ] k )t
(3)
(4)
0
e
t
e
1
~1
and
Isomerization procedure
K
\ k /k
eq
1
~1
b-ODAP (1.00 mg) was dissolved in 5.00 mL of bu†er or dis-
tilled water in a tube and kept in a thermostated water bath.
Periodically 0.30 ml of solution was injected into the CZE
system directly. The sampling intervals from the tube were 60,
20 and 5 min at temperatures 5, 75 and 95 ¡C, respectively.
The temperature variation of the thermostated water bath was
within ^0.1 ¡C.
where K is the equilibrium constant, the subscripts 0 and t
indicate concentrations at the beginning of the experiment
eq
and at time t, respectively. A plot of ln(x [ x )/(x [ x ) vs. t
0
1
e
t
e
thus yields the sum of the rate constant k ] k , and by eqn.
~1
(4), the rate constants k and k could be calculated.
~1
1
Rate constants k and k are determined at pH \ 3.0, 6.5
1
~1
and 11.0 at 55 ¡C, pH \ 6.5 at 75 ¡C and pH \ 6.5 at 95 ¡C
respectively. Fig. 2 shows the data obtained from the experi-
ment at 55 ¡C, the computation results are shown in Table 1.
Results and discussion
The activation energy E and pre-exponential factor A are
calculated by the equation of Arrhenius,
a
InÑuence of pH on the equilibrium concentration ratio of a- and
b-ODAP
A
E
B
a
k \ A exp [
(5)
(6)
RT
b-ODAP in the bu†er solutions in the range from pH \ 2.5 to
pH \ 13.0 was kept in a thermostated water bath for 24 h at
T \ 75 ¡C, the concentration ratio obtained can be regarded
as the equilibrium concentration ratio. The results are shown
in Fig. 1. They indicate that the equilibrium concentration
ratio of a-ODAP decreased with the increase of pH.
or
E
RT
a
ln k \ [
] ln A
where k is the rate constant, R is the gas constant, and T is
the temperature in Kelvin. E and A are obtained from the Ðt
a
of the equation for the plot of ln k vs. 1/RT (Fig. 3). The
Kinetic parameters
computation results at pH \ 6.5 were respectively: E \ 111
al
The reaction of thermal isomerization of b-ODAP is recipro-
cal.
kJ mol~1, A \ 2.97 ] 1012 s~1 for the reaction from
1
b-ODAP to a-ODAP, and E
] 1012 s~1 for the reaction from a-ODAP to b-ODAP.
\ 110 kJ mol~1, A \ 3.93
a~1
~1
Fig. 1 Comparison of the equilibrium concentration ratio of
a-ODAP at di†erent values of pH.
Fig. 2 Plot of ln(x [ x )/(x [ x ) vs. t at temperature 55 ¡C,
t
0
e
e
pH \ 3.0, pH \ 6.5 and pH \ 11.0.
Table 1 Rate constants of thermal isomerization of b-ODAP
Equilibrium
concentration ratio
of a-ODAP (%)
Conditions
K
k ] k /10~5 s~1
~1
k /10~5 s~1
k
/10~5 s~1
eq
1
1
~1
55 ¡C (pH \ 3.0)
55 ¡C (pH \ 6.5)
55 ¡C (pH \ 11.0)
75 ¡C (pH \ 6.5)
95 ¡C (pH \ 6.5)
34.5
31.0
28.6
32.5
32.3
0.527
0.449
0.401
0.481
0.477
1.70
1.70
1.88
19.3
138
0.59
0.53
0.54
6.27
44.6
1.11
1.17
1.34
13.0
93.4
3772
Phys. Chem. Chem. Phys., 1999, 1, 3771È3773

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of a-ODAP comes to only 18% after reaction for 96 hours. So
they concluded that the isomerization reaction at pH 6.6 was
considerably slower. However, from Fig. 4, it could be seen
that the equilibrium of the reaction at three di†erent pH
values was reached nearly at the same time, with di†erent
equilibrium ratios, respectively. Based on these considerations,
we think the experimental results are probably caused by not
only kinetic reasons but mainly thermodynamics.
Acknowledgements
This project is Ðnancially supported by the National Natural
Science Foundation (No. 39770469 and No. 29475194),
Natural Science Foundation of Gansu Province.
Fig. 3 Plot of ln k vs. 1/T (K~1): (>) indicates reaction from
b-ODAP to a-ODAP, (@) indicates reaction from a-ODAP to
b-ODAP.
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Eqn. (3) could be converted into
(x [ x )/(x [ x ) \ exp[(k ] k )t]
~1
(7)
(8)
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Paper 9/02229E
Fig. 4 Experimental (points) and simulated (curves) increase of con-
centration ratio of a-ODAP at 55 ¡C in di†erent pH.
Phys. Chem. Chem. Phys., 1999, 1, 3771È3773
3773