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Electrochemical studies of the decomposition of thiazolidine-4-carboxylic acids in aqueous solutions
129
J. Electroanal. Chem, 288 (1990) 129-141 Elsevier Sequoia S.A., Lausamre
Electrochemical studies of the decomposition of thiazolidine-4-carboxylic acids in aqueous solutions Zenon J. Karpiuski, Marta Kamy and Zenon Kublii Department
of Chemistry,
University of Warsaw, Pasteura 1, 02093 Warsaw (Poland)
(Received 25 June 1989; in revised form 2 March 1990)
ABSTRACT The decomposition reactions and interactions with mercury ions of thiazolidin~4-carboxylic acid (Thz) and its 2-substituted derivatives 2-propyl- (PrThz) and 2-Garabino-tetrahydroxybutyl-thiazolidme4-carboxylic (AT&) acids, in aqueous solutions at pH from 4.5 to 8, have been studied by cyclic voltammetry and by normal pulse and Osteryoung square wave voltammetry. Thiazolidines form unstable complexes with Hg(I) ions which transform into mercaptides. Thz and its 2-substituted derivatives decompose in nearly neutral aqueous solutions, liberating L-cysteine. The rate of the decomposition depends distinctly on the nature of the C-2 substituent and on the solution pH. It is the highest for the alkyl-substituted Thzs and exhibits a maximum at pH near 6. A significant change of the decomposition rate occurs near physiological pH. This indicates that the non-enzymatic liberation of L-cysteine in vivo may be induced by changes in pH. The correlation between the decomposition rate of Thz derivatives and literature data on their protective effect against hepatoxicity confirms that they act as prodrugs of L-cysteine. The presence of oxygen accelerates the decomposition of Thz and its derivatives distinctly.
INTRODUCTION
The importance of thiazolidine-4-carboxylic acid (thioproline, Thz) and its 2-substituted derivatives in biological and medicinal chemistry is well documented. These compounds are formed in reactions of aldehydes with L-cysteine and appear among mammalian metabolites [l-5]. There have been extensive studies of such compounds in many pharmaceutical applications [6-111. In particular, detailed investigations of their protective action against hepatoxicity have been reported recently [12-141. Biological functions of Thz derivatives have been attributed repeatedly to their ring-opening reactions, exposing the reactive sulfhydryl group and ultimately liberating L-cysteine. There are, however, very little data on the decomposition reactions of Thz derivatives in aqueous solutions. Riemschneider and Hoyer [15]
130
reported results of qualitative experiments and polarimetric measurements for a number of aliphatic and aromatic substituted Thzs. The decomposition rate constants were estimated only for 2,2dialkyl derivatives. Pesek and Frost initially observed indications of the decomposition of 2-a&l-substituted Thz only in strongly basic solutions [16]. Recently, however, they determined the pH rate of the decomposition profile for 2,2,5,5-tetramethyl Thz at pH from 1 to 8 [17]. Recent studies of the voltammetric behaviour of Thz and its derivatives on mercury electrodes have reported anodic waves due to reactions of these compounds with mercury ions [18,19]. The anodic waves appear at potentials less negative than the anodic waves of compounds with thiol groups, thus allowing simultaneous detection of Thz derivatives and their decomposition products containing sulphydryl groups. Besides good monitoring of the decomposition reaction, the methodology of electroanalytical experiments enables observations of the effect of oxygen on the decomposition of Thz derivatives. The latter effect was not reported in the previous spectroscopic studies. Interactions of Thz derivatives with mercury ions are also of interest, since they indicate the reactivity of these compounds toward heavy metal ions. It has been reported that Hg(I1) [20] and Ag(I) [21] ions accelerate the decomposition of Thz and some of its derivatives. Electrochemical studies of the stability of Thz and its 2-substituted derivatives, 2-propyl-thiazolidine-4-carboxylic acid (PrThz) and 2-L-arabino-tetrahydroxybutylthiazolidine-4-carboxylic acid (Al%@, at biologically important pHs of aqueous solutions, in the absence and presence of oxygen, are reported in this work. Further, the mechanism of the reaction of these compounds with mercury ions is discussed. EXPERIMENTAL
Electrochemical experiments were mostly performed with a Bioanalytical Systems BAS-100 Electrochemical Analyzer. In some experiments a Radelkis OH-105 polarograph was used. A saturated calomel electrode served as reference electrode in a conventional three-electrode cell and all potentials are referred to this electrode. As a working electrode a SMDE (Laboratorni Pristroje) with a mercury drop area of 0.0167 cm2 was used. In the kinetic measurements, normal pulse (NP) voltammetry at 50 ms pulse width and square wave voltammetry according to Osteryoung and Osteryoung [22] (OSW voltammetry) at a frequency of 10 Hz and a square wave amplitude of 25 mV were used. All experiments were performed in a water-jacketed cell and the water temperature was maintained at 298 f 0.2 K. Thiazolidine-4-carboxylic acid and its derivatives were synthesized from L-cysteine and the appropriate aldehyde according to the procedure described elsewhere [23]. Stock solutions of Thz derivatives (1 to 5 mM) were prepared freshly by dissolving weighed samples of these compounds in deaerated solutions of 0.1 M Na,HPO, or 0.05 M CH,COOH and kept under argon. The decomposition of Thz and its derivatives was much slower at pH ca. 3 and ca. 8 than at pH from 5 to 7 (see below). All buffer solutions were prepared from reagent grade chemicals and
131
triply distilled water. The solutions studied were deaerated carefully and maintained under argon throughout all experiments. REmJLT!s
Interactions with mercury ions The cyclic voltammograms in Fig. 1 show characteristic features of the electrochemical behaviour of Thz derivatives at mercury electrodes. An anodic peak (ai) appearing during the first potential sweep is followed by two cathodic peaks which are distinctly dependent on the time of the experiment, i.e. on the potential scan rate. At a fast scan rate of 50 V/s a nearly reversible pair of peaks (a,, cl) was observed for PrThz (Fig. 1, curve 1). No additional peak was evident during the second potential scan. At a slow scan rate of 0.5 V/s, however, the cathodic peak c1 was not observed, but the products of the anodic reaction were reduced in peak c2, and an additional anodic peak a2, forming a nearly reversible couple with peak ca, appearing during the second sweep toward positive potentials (Fig. 1, curve 3). In the intermediate range of potential scan rates all four peaks could be observed (Fig. 1, curve 2). Such behaviour indicated chemical transformations of the Thz derivatives in their reactions with mercury ions. The unstable oxidation products formed
I
0.0
I
-0.2
I
-O.’
E /
V
Fig. 1. Effect of potential scan rate on the cyclic voltammogmm of 2.5 X 10e5 M PrThz. u: (1) 50, (2) 5, (3) 0.5 V/s. Phosphate buffer, pH 7.4. (- - -) Second scan. Starting potential -0.6 V.
132
I
0.0
I
I
I
-0.2
-0.4
-0.6
E/V
Fig. 2. Cyclic voltammograms of 2 X 10e4 M AThz (1, 4), 1 X 10e5 M Cys (2), and of a mixture of both compounds (3). U: (l-3) 0.5, (4) 0.1 V/s. Phosphate buffer, pH 7.4.
initially were transformed into the final products of the reactions between Thz derivatives and mercury ions. Changes in the structure of the organic molecules were reflected in the differences of the potentials of the initial redox couple (peaks al/cl) and the transformation products (peaks a2/c2). Cyclic voltammetric experiments indicated similar oxidation mechanisms for all the Thz derivatives studied. However, more complex cyclic voltammograms were observed for AThz (Fig. 2). The initial cathodic peak c1 was much broader than those for TIu and PrThz and its shape and position depended on the potential scan rate. This potential dependence probably reflects time-dependent transformations of an initial oxidation product. There are several coordination sites in the molecule of the sugar derivative that could interact with mercury ions. Additionally, different orientations of the adsorbed complex with the polar ligand would affect the capacitive contribution to the observed current. Besides the initial and the secondary peak couples, voltammogram 1 shows a more positive anodic peak. Similar peaks were observed for all Thz derivatives at high concentrations and/or at low potential scan rates, after formation of amounts of the oxidation products corresponding to more than a monolayer coverage of the electrode surface (Fig. 1, curve 3). These additional peaks reflect the formation of additional products on a mercury electrode fully covered by the oxidation products. Cyclic voltammograms with similar secondary effects have been reported for Thz [18]. These heterogeneous surface processes are of minor impor-
133
tance for the biological functions of Thz derivatives and have not been studied in this work. On the other hand, the most important feature of the voltammograms obtained for AThz was a clear reflection of the transformations of the mercaptides formed by the reaction of AThz with mercury ions. The secondary cathodic-anodic peak couple, a2/c2 (Fig. 2, curve l), appeared at potentials slightly more negative than the voltammetric peaks observed in an L-cysteine solution (curve 2) and in a solution containing both these compounds, two overlapping anodic and better resolvable cathodic peaks were evident (curve 3). This shows that the peaks a2/c2 reflect the reduction and formation of compounds of mercury with products of the thiazolidme ring-opening reaction of AThz. At very low scan rates still another cathodic peak was observed for AThz (Fig. 2, curve 4). This peak, appearing at potentials characteristic for L-cysteine, indicated further transformations of the mercury compounds, forming the cysteine compounds. The shapes of scan rate dependences of both main cathodic peaks (cr and c;?) for PrThz indicated their surface character. On the other hand, the height of anodic peak aI followed a square root dependence, indicating its diffusional control, up to scan rates near 1 V/s, and then the peak height increased faster until at 50 V/s the anodic peak shape showed clearly effects of adsorption of Thz derivatives at the mercury electrode surface (Fig. 1, curve 1). The cyclic voltammetric experiments revealed the complex behaviour of Thz derivatives at mercury electrodes. Besides reflections of the chemical transformations of the oxidation products, they exhibited secondary effects due to accumulation of the oxidation products at the electrode surface. Such effects of product accumulation and transformation are least pronounced in normal pulse (NP) voltammetric experiments [19,24], and the latter technique was used to study the initial steps in the reaction of Thz derivatives with mercury ions, particularly in attempts to determine whether mercurous or mercuric ions are involved in these reactions. Undisturbed NP voltammograms could be obtained at concentrations of Thz derivatives, chosen such that the amounts of oxidation products formed during the pulse time were limited to ca. 10% of the monolayer electrode coverage [19]. Under these conditions one well developed anodic NP voltammetric wave was obtained for each Thz derivative studied (Fig. 3). The NP waves for PrThz exhibited diffusion controlled limiting current, as was indicated by their pulse width dependence. The half-wave potential of the PrThz waves was independent of the pulse width and PrThz concentration within wide ranges (pulse widths from 3 to 200 ms, and concentrations from 1 to 500 PM were used). This indicates a reversible oxidation process with little effects of product accumulation and transformation. Also, log{ i/( i, - i)} vs. E plots for the NP waves were linear, although their slopes depended to some extent on the pulse width and PrThz concentration. Most of the waves exhibited slopes between 50 and 60 mV, with the lower values observed for smaller pulse widths and lower PrThz concentrations. These slope variations may result from the effects of adsorption of PrThz molecules on the electrode surface. The effects of reactant adsorption were even more pronounced for AThz, for which, at lower concentrations and/or shorter pulse widths, NP waves with a distinct
134 I
I
’
&
OT
1
0.1
2
0
-O.l_
_
-_I
GJJ a
=3
‘-
0
-0.1
--- I'
0.1 -
3
O-
0 L
-O.l-A I -0.1
-0.2
I -4 -0.3
E/V
Fig. 3. Effect of pulse width on normal pulse voltammograms of 1 x 10m4 M PrThz. t,/ms the curves. Phosphate buffer, pH 7.4.
/ -".5E / v
indicated on
Fig. 4. Changes in the cyclic voltammogram upon storage of 5 x lo-’ M PrThz in phosphate buffer, pH 6.0. Time after solution preparation: (1) 0, (2) 1, (3) 6 h. v = 16 mV/s.
maximum were observed. Such behaviour indicates stronger adsorption of the reactant than of the product of the electrode process [25,26]. The NP waves of Thz and AThz oxidation exhibited a pulse width dependence of their half-wave potentials, which shifted to less negative values at shorter pulse widths. This may be attributed to effects of the follow-up transformations of the oxidation products. In spite of the additional effects of adsorption and follow-up chemical transformations, the NP results indicate interactions of the Thz derivatives with Hg(1) ions as the initial step in the anodic process. Also, the width of the anodic peaks observed in OSW voltammetric experiments, close to 120 mV, indicates a one-electron oxidation process. The follow-up transformations evident in the cyclic voltammetric experiments reflect the instability of the initially formed complexes of Thz derivatives with Hg(1) which transform into mercaptides. This was indicated by the potentials of the peaks $/a,, which appeared in a region characteristic for compounds with sulphydryl groups. In spite of difficulties in discriminating between mercurous cysteinate and compounds of mercury with N-substituted cysteine and/or with the respective Schiff bases, some indications of the initial formation of the latter compounds were observed. Thus, there were differences between the shapes and potentials of the c, peaks for different Thz derivatives, and for AThz the c/a,
135
peak couple appeared at potentials more negative than the peaks of L-cysteine (Fig. 2). Decomposition reactions Thiazolidine ring-opening reactions were observed not only after interactions of Thz and its derivatives with Hg(1) ions. Aqueous solutions of the compounds studied were also unstable in the absence of mercury ions. This was reflected in changes in the results of electrochemical experiments observed after storing solutions of Thz and its derivatives for the prolonged times. The cyclic voltammograms in Fig. 4 show a diminishing of the original anodic peak due to PrThz and the simultaneous appearance and increase of an additional peak at more negative potentials. Six hours after preparing the solution, all PrThz had decomposed and only the more negative anodic peak was observed. The last cyclic voltammogram in Fig. 4 is identical to that obtained in a solution of L-cysteine. Also, OSW voltammograms recorded in a solution of the decomposition products exhibited the same shapes of the forward and reverse currents and the peak potentials as those for an L-cysteine solution (Fig. 5). These results show that during the decomposition of
-0.1
-0.3
-0.5 E/V
-0.5
E/V
-1.0
Fig. 5. OSW voltammograms of the decomposition products of PrThz (1) and L-cysteine (2). Phosphate buffer, pH 6.0. (- - -_) Forward and reverse currents, () net current. Fig. 6. Sampled dc polarograms of L-cyst&e (1) and PrTbz (2) in air-saturated phosphate buffer, pH 6.0. Drop and sampling times 1 and 0.02 s. Time (in min) after solution preparation indicated on the curves.
136
COOH
/-I
HS
0 NH2
+
R&H
Scheme 1.
Thz derivatives in aqueous solutions, L-cysteine is liberated and can be detected and identified by electroanalytical techniques. On the other hand, intermediate products of the decomposition of Thz derivatives (Scheme 1) could not be detected in such experiments. In freshly prepared solutions of Thz and its derivatives only one anodic wave was observed, and no open-ring intermediates could be detected, even in the absence of cysteine. Also, during the decomposition no additional wave appeared. This indicates distinctly lower concentrations of the intermediates than of L-cysteine. At least for AThz the potential difference between the anodic and cathodic peaks due to cysteine and the open-ring intermediates (see above) would have allowed the detection of intermediates at concentrations comparable to that of the amino acid. Simultaneous monitoring of the changes in the concentrations of Thz derivatives and L-cysteine enabled kinetic analysis of the decomposition reaction. Two pulse voltammetric techniques were used in such experiments. OSW voltammetry exhibited the virtues of speed and better identification through the shapes of the forward and reverse currents as well as the peak potentials, while suffering from decreased accuracy of the measurements for overlapping peaks. On the other hand, the additive waves observed in the NP voltammetric experiments enabled more accurate measurements of the changing concentrations of Thz derivatives and L-cysteine. The long-time experiments were tested additionally for effects of the presence of oxygen on the reactions studied. It was found that the decomposition rate of Thz and its derivatives is distinctly higher in the presence of oxygen (see below). The sampled dc polarograms in Fig. 6-1 show the oxidation of cysteine by oxygen. The anodic cysteine wave decreased in time and a cathodic wave, due to cystine reduction, appeared and gradually increased. DC polarograms recorded in PrThz solution in the presence of oxygen (Fig. 6-2) also show the formation of cystine. Since cystine may be formed not only by cysteine oxidation but also in reactions of oxygen with intermediate products of the decomposition of Thz derivatives, the kinetics of the decomposition reaction were studied in carefully deaerated solutions. Examples of typical pulse voltammetric curves obtained in studies of the kinetics of the decomposition of Thz derivatives are shown in Fig. 7. Distinct changes in the
137
1o-3t/s Fig. 7. OSW (1) and NP (2) voltammograms of PrTbz in phosphate buffer, pH 6.0. Time (in min) after solution preparation indicated on the curves. Fig. 8. Kinetic analysis of the decomposition of PrThz at pH 6.0 (1) in the absence and (2) in the presence of oxygen.
pulse voltammetric curves, observed after storing the PrThz solution, reflect the decomposition of the Thz derivatives and the formation of the reaction product L-cysteine. Apparent differences between the relative changes of the heights of the OSW voltammetric peaks and the NP voltammetric waves result from differences in their concentration dependences, i.e. in their sensitivities for Thz derivatives and cysteine. The net current in OSW voltammetry, the difference between the forward and reverse currents, is larger than either of the contributions for reversible systems, such as cysteine/cystine (Fig. 5). On the other hand, the chemical irreversibility of the oxidation of Thz derivatives (see above) causes a significant decrease of the OSW voltammetric net current, which, in such cases, is smaller than the forward current. Diffusion controlled limiting currents in NP voltammetry are independent of follow-up chemical transformations and the slight differences between the sensitivities observed for Thz derivatives and cysteine result solely from differences between the respective diffusion coefficients. In order to control the decomposition reactions better, the kinetic analysis was performed in terms of time dependences of the concentrations, rather than of the directly measured electroanalytical signals. Using linear calibration plots determined for Thz and its derivatives and for cysteine at each pH studied (small but distinct pH dependences of the respective sensitivities were observed for cysteine as well as Thz derivatives), the concentration vs. time dependences were analysed. Throughout the decomposition reaction, the sum of the concentrations of Thz derivative and L-cysteine was equal to the initial concentration of the former compound. The latter relation was used as an additional
138
test of the accuracy of the measurements of the varying concentrations and yielded relative standard deviations below 5%. The variations of the concentrations of PrThz during the decomposition reactions followed pseudo-first order kinetics. Linear regression for the dependence In c = k,t + In c0 for PrThz at pHs from 4.5 to 7.4 yielded correlation coefficients better than 0.99 (Fig. 8, curve 1). The slopes of this dependence afforded values of the apparent decomposition rate constant (k,). The k, values obtained for PrThz decomposition from results of NP and OSW voltammetric experiments agreed within 5% and the relative standard deviations of the individual linear regression were below 10%. Determinations of the decomposition rate constants for Thz and AThz were less accurate. This was caused by their much slower decomposition, resulting in small changes in the concentration of Thz derivatives. During an average 8 h long experiment, the decrease in Thz or AThz concentration amounted to only ca. 10% of the initial concentration. An additional difficulty impeding studies of AThz decomposition was caused by a small difference between the half-wave and peak potentials for AThz and L-cysteine (Fig. 2). Therefore, the relative standard deviations for k, determinations under these conditions reached 30%. The adherence of the decomposition reaction to a first-order rate law was tested additionally in experiments performed at different initial concentration of PrThz, from 1 x 10e5 to 1 X 1O-3 M. Experiments at higher concentrations of PrThz were performed in a different way to check the decomposition reaction for a possible dependence on the presence of small amounts of metallic mercury, which was difficult to avoid in the usual pulse voltammetric experiments. In these experiments the decomposition reaction proceeded outside the electrochemical cell and samples of the reaction mixture were analysed immediately. The decomposition rate constant was found to be independent of the presence of small amounts of mercury and of the initial PrThz concentration, except for its highest value. The k, value obtained at an initial concentration of PrThz equal to 1 X 10e3 M was almost a factor of two lower than at low concentrations of the Thz derivatives. This may indicate a change in the decomposition kinetics at high concentrations of Thz derivative, which are, however, of less biological significance. The mean values of the decomposition rate constants of Thz derivatives are collected in Table 1 and show the distinct dependence of the decomposition kinetics on pH and on the nature of the substituent at the C-2 position of Thz. In addition to kinetic measurements of the decomposition of Thz derivatives in carefully deaerated solutions, monitoring of the decomposition reactions in the presence of oxygen was performed in sampled dc polarographic experiments. A change in the mechanism and kinetics of the decomposition reaction was observed: a distinct cathodic wave reflected the formation of cystine (Fig. 6, curve 2). In spite of a different decomposition mechanism, however, the time dependence of the decreasing concentration of PrThz at pH 6.0 followed a pseudo-first-order rate law with correlation coefficient 0.993 (Fig. 8, curve 2). The apparent decomposition rate constant in the presence of oxygen was nearly a factor of two larger than in the deaerated solution. The slower decomposition reactions of PrThz at pH 7.4 and of Thz at pH 6.0 exhibited more complex time dependences. Only during the initial 3 h
139
TABLE 1 First-order rate constants for the decomposition of Thz and its derivatives Compound
PH
Thz
7.4 6.0 6.0+4
10’ k,/s
a
0.06 0.3 1.3
PrTbz
8.0 7.4 7.4+0* a 6.7 6.0 6.0+4 ’ 5.2 4.5
0.03 1.6 2.3 4.3 7.3 14.8 4.7 2.1
ATlIZ
7.4 6.0
0.08 0.3
a Air-saturated solution.
were In c vs. t plots linear, yielding k, values distinctly higher than in the absence of oxygen (Table 1). In experiments on longer time scales an additional acceleration of the decomposition reactions was observed. DISCUSSION
Electrochemical studies of the reactions of Thz derivatives in aqueous solutions showed the importance of the ring-opening reaction in the chemistry of these compounds. This reaction follows the initial formation of unstable complexes of Thz derivatives with Hg(1). It is also the initial step in the decomposition reaction of Thz derivatives in aqueous solutions. Fast scan cyclic voltammetric and pulse voltammetric experiments revealed the initial steps in the anodic processes of Thz and its derivatives at mercury electrodes. The formation of unstable complexes of Thz derivatives with Hg(1) ions is followed by the opening reaction of the thiazolidine ring in which mercaptides are formed. Further transformations of the mercury compounds lead to the formation of cysteine. These findings differ from the assumptions of Costa Garcia and Tunon Blanco [18], who, using slow scan cyclic voltammetric and cathodic stripping experiments, did not observe the initial oxidation products. Therefore, they assumed that, in the anodic reaction of Thz at the mercury electrode, L-cysteine is formed and reacts with mercury ions. Electroanalytical studies of aqueous solutions of Thz and its derivatives showed their decomposition, in which L-cysteine is liberated. No open-ring intermediates of the decomposition were detected either in freshly prepared solutions of the Thz derivatives studied or during their decomposition. The detection limits of the pulse voltammetric methods used would have allowed observation of compounds with sulphydryl groups in concentrations as low as 1 PM. Thus, the results indicate
140
rather low equilibrium concentrations of the respective Schiff bases, which were not observed even in freshly prepared solutions containing no cysteine. The proximity of the anodic waves of various compounds with sulphydryl groups hindered measurements of the open-ring decomposition intermediates (Scheme 1) in distinctly lower concentrations. Still, a detection of these compounds in concentrations comparable to that of cysteine should be possible, at least for AThz. For Thz and PrThz, the structural differences between the open-ring intermediates and L-cysteine are much smaller and the transformations of the mercaptides are probably faster than for the sugar derivative. Therefore it is difficult to judge which of these two reasons precluded detection of the intermediate mercaptides. Because of these difficulties, it is impossible to estimate the positions of the anodic signals due to the open-ring intermediates of the decomposition of Thz and PrThz. However, it seems probable that not only for AThz, but also for the other Thz derivatives the decomposition intermediates appear in low concentrations. This indicates the relative instability of the intermediates and suggest that the thiazolidine ring-opening reaction is the rate determining step in the decomposition of Thz and its derivatives studied. Kinetic measurements of the rate of decomposition of Thz and its derivatives in aqueous solutions showed the distinct dependence of the decomposition rate constant on the nature of the C-2 substituent and on pH. These findings may aid in understanding the biological functions of the compounds studied. Formation and decomposition reactions of thioproline derivatives have been reported to be involved in several biochemical cycles and their function as latent L-cysteine, liberating this important amino acid intracellularly, has often been assumed. In this perspective the observed significant dependence of the decomposition rate of Thz derivatives around physiological pH is particularly interesting. It indicates that non-enzymatic liberation of L-cysteine in vivo may be induced by changes in pH. A pH dependence of the decomposition of 2,2-dialkyl- [15] and 2,2,5,5_tetramethyl- [17] Thz has been observed in previous studies. There is a discrepancy, however, between these reports concerning pHs above 7. Pesek and Frost [17] reported an increase in the decomposition rate at pHs higher than 7, while Riemschneider and Hoyer [15] observed a distinctly slower decomposition at pH 8.8 than at pH 6.8. These investigations of particularly unstable Thz derivatives, performed at high concentrations and apparently in the presence of oxygen, are difficult to compare with the present work, concentrated on studies of Thz and its 2-substituted derivatives representing two groups of prodrugs of L-cysteine used by Nagasawa and co-workers [12-141, and performed at low concentrations and in the absence of oxygen. The effect of the nature of the C-2 substituent on the rate of decomposition correlates clearly with the relative efficacies of Thz derivatives in their protection against the hepatoxic action of acetaminophen [12-141. PrThz has been reported to be much more liver-protective than Thz, which exhibited an efficacy similar to that of AThz. This correlation supports the hypothesis of Nagasawa and co-workers [12-141, who assumed that the intracellular non-enzymatic liberation of L-cysteine causes the hepatoxicity protection by supplying this amino acid precursor of glutathione to the cell.
141 ACKNOWLEDGEMENTS
We thank Jan Radomski for preparation of the Thz derivatives. This work was supported financially by the CPBP.Ol.15 research program. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
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J. Electroanal. Chem, 288 (1990) 129-141 Elsevier Sequoia S.A., Lausamre
Electrochemical studies of the decomposition of thiazolidine-4-carboxylic acids in aqueous solutions Zenon J. Karpiuski, Marta Kamy and Zenon Kublii Department
of Chemistry,
University of Warsaw, Pasteura 1, 02093 Warsaw (Poland)
(Received 25 June 1989; in revised form 2 March 1990)
ABSTRACT The decomposition reactions and interactions with mercury ions of thiazolidin~4-carboxylic acid (Thz) and its 2-substituted derivatives 2-propyl- (PrThz) and 2-Garabino-tetrahydroxybutyl-thiazolidme4-carboxylic (AT&) acids, in aqueous solutions at pH from 4.5 to 8, have been studied by cyclic voltammetry and by normal pulse and Osteryoung square wave voltammetry. Thiazolidines form unstable complexes with Hg(I) ions which transform into mercaptides. Thz and its 2-substituted derivatives decompose in nearly neutral aqueous solutions, liberating L-cysteine. The rate of the decomposition depends distinctly on the nature of the C-2 substituent and on the solution pH. It is the highest for the alkyl-substituted Thzs and exhibits a maximum at pH near 6. A significant change of the decomposition rate occurs near physiological pH. This indicates that the non-enzymatic liberation of L-cysteine in vivo may be induced by changes in pH. The correlation between the decomposition rate of Thz derivatives and literature data on their protective effect against hepatoxicity confirms that they act as prodrugs of L-cysteine. The presence of oxygen accelerates the decomposition of Thz and its derivatives distinctly.
INTRODUCTION
The importance of thiazolidine-4-carboxylic acid (thioproline, Thz) and its 2-substituted derivatives in biological and medicinal chemistry is well documented. These compounds are formed in reactions of aldehydes with L-cysteine and appear among mammalian metabolites [l-5]. There have been extensive studies of such compounds in many pharmaceutical applications [6-111. In particular, detailed investigations of their protective action against hepatoxicity have been reported recently [12-141. Biological functions of Thz derivatives have been attributed repeatedly to their ring-opening reactions, exposing the reactive sulfhydryl group and ultimately liberating L-cysteine. There are, however, very little data on the decomposition reactions of Thz derivatives in aqueous solutions. Riemschneider and Hoyer [15]
130
reported results of qualitative experiments and polarimetric measurements for a number of aliphatic and aromatic substituted Thzs. The decomposition rate constants were estimated only for 2,2dialkyl derivatives. Pesek and Frost initially observed indications of the decomposition of 2-a&l-substituted Thz only in strongly basic solutions [16]. Recently, however, they determined the pH rate of the decomposition profile for 2,2,5,5-tetramethyl Thz at pH from 1 to 8 [17]. Recent studies of the voltammetric behaviour of Thz and its derivatives on mercury electrodes have reported anodic waves due to reactions of these compounds with mercury ions [18,19]. The anodic waves appear at potentials less negative than the anodic waves of compounds with thiol groups, thus allowing simultaneous detection of Thz derivatives and their decomposition products containing sulphydryl groups. Besides good monitoring of the decomposition reaction, the methodology of electroanalytical experiments enables observations of the effect of oxygen on the decomposition of Thz derivatives. The latter effect was not reported in the previous spectroscopic studies. Interactions of Thz derivatives with mercury ions are also of interest, since they indicate the reactivity of these compounds toward heavy metal ions. It has been reported that Hg(I1) [20] and Ag(I) [21] ions accelerate the decomposition of Thz and some of its derivatives. Electrochemical studies of the stability of Thz and its 2-substituted derivatives, 2-propyl-thiazolidine-4-carboxylic acid (PrThz) and 2-L-arabino-tetrahydroxybutylthiazolidine-4-carboxylic acid (Al%@, at biologically important pHs of aqueous solutions, in the absence and presence of oxygen, are reported in this work. Further, the mechanism of the reaction of these compounds with mercury ions is discussed. EXPERIMENTAL
Electrochemical experiments were mostly performed with a Bioanalytical Systems BAS-100 Electrochemical Analyzer. In some experiments a Radelkis OH-105 polarograph was used. A saturated calomel electrode served as reference electrode in a conventional three-electrode cell and all potentials are referred to this electrode. As a working electrode a SMDE (Laboratorni Pristroje) with a mercury drop area of 0.0167 cm2 was used. In the kinetic measurements, normal pulse (NP) voltammetry at 50 ms pulse width and square wave voltammetry according to Osteryoung and Osteryoung [22] (OSW voltammetry) at a frequency of 10 Hz and a square wave amplitude of 25 mV were used. All experiments were performed in a water-jacketed cell and the water temperature was maintained at 298 f 0.2 K. Thiazolidine-4-carboxylic acid and its derivatives were synthesized from L-cysteine and the appropriate aldehyde according to the procedure described elsewhere [23]. Stock solutions of Thz derivatives (1 to 5 mM) were prepared freshly by dissolving weighed samples of these compounds in deaerated solutions of 0.1 M Na,HPO, or 0.05 M CH,COOH and kept under argon. The decomposition of Thz and its derivatives was much slower at pH ca. 3 and ca. 8 than at pH from 5 to 7 (see below). All buffer solutions were prepared from reagent grade chemicals and
131
triply distilled water. The solutions studied were deaerated carefully and maintained under argon throughout all experiments. REmJLT!s
Interactions with mercury ions The cyclic voltammograms in Fig. 1 show characteristic features of the electrochemical behaviour of Thz derivatives at mercury electrodes. An anodic peak (ai) appearing during the first potential sweep is followed by two cathodic peaks which are distinctly dependent on the time of the experiment, i.e. on the potential scan rate. At a fast scan rate of 50 V/s a nearly reversible pair of peaks (a,, cl) was observed for PrThz (Fig. 1, curve 1). No additional peak was evident during the second potential scan. At a slow scan rate of 0.5 V/s, however, the cathodic peak c1 was not observed, but the products of the anodic reaction were reduced in peak c2, and an additional anodic peak a2, forming a nearly reversible couple with peak ca, appearing during the second sweep toward positive potentials (Fig. 1, curve 3). In the intermediate range of potential scan rates all four peaks could be observed (Fig. 1, curve 2). Such behaviour indicated chemical transformations of the Thz derivatives in their reactions with mercury ions. The unstable oxidation products formed
I
0.0
I
-0.2
I
-O.’
E /
V
Fig. 1. Effect of potential scan rate on the cyclic voltammogmm of 2.5 X 10e5 M PrThz. u: (1) 50, (2) 5, (3) 0.5 V/s. Phosphate buffer, pH 7.4. (- - -) Second scan. Starting potential -0.6 V.
132
I
0.0
I
I
I
-0.2
-0.4
-0.6
E/V
Fig. 2. Cyclic voltammograms of 2 X 10e4 M AThz (1, 4), 1 X 10e5 M Cys (2), and of a mixture of both compounds (3). U: (l-3) 0.5, (4) 0.1 V/s. Phosphate buffer, pH 7.4.
initially were transformed into the final products of the reactions between Thz derivatives and mercury ions. Changes in the structure of the organic molecules were reflected in the differences of the potentials of the initial redox couple (peaks al/cl) and the transformation products (peaks a2/c2). Cyclic voltammetric experiments indicated similar oxidation mechanisms for all the Thz derivatives studied. However, more complex cyclic voltammograms were observed for AThz (Fig. 2). The initial cathodic peak c1 was much broader than those for TIu and PrThz and its shape and position depended on the potential scan rate. This potential dependence probably reflects time-dependent transformations of an initial oxidation product. There are several coordination sites in the molecule of the sugar derivative that could interact with mercury ions. Additionally, different orientations of the adsorbed complex with the polar ligand would affect the capacitive contribution to the observed current. Besides the initial and the secondary peak couples, voltammogram 1 shows a more positive anodic peak. Similar peaks were observed for all Thz derivatives at high concentrations and/or at low potential scan rates, after formation of amounts of the oxidation products corresponding to more than a monolayer coverage of the electrode surface (Fig. 1, curve 3). These additional peaks reflect the formation of additional products on a mercury electrode fully covered by the oxidation products. Cyclic voltammograms with similar secondary effects have been reported for Thz [18]. These heterogeneous surface processes are of minor impor-
133
tance for the biological functions of Thz derivatives and have not been studied in this work. On the other hand, the most important feature of the voltammograms obtained for AThz was a clear reflection of the transformations of the mercaptides formed by the reaction of AThz with mercury ions. The secondary cathodic-anodic peak couple, a2/c2 (Fig. 2, curve l), appeared at potentials slightly more negative than the voltammetric peaks observed in an L-cysteine solution (curve 2) and in a solution containing both these compounds, two overlapping anodic and better resolvable cathodic peaks were evident (curve 3). This shows that the peaks a2/c2 reflect the reduction and formation of compounds of mercury with products of the thiazolidme ring-opening reaction of AThz. At very low scan rates still another cathodic peak was observed for AThz (Fig. 2, curve 4). This peak, appearing at potentials characteristic for L-cysteine, indicated further transformations of the mercury compounds, forming the cysteine compounds. The shapes of scan rate dependences of both main cathodic peaks (cr and c;?) for PrThz indicated their surface character. On the other hand, the height of anodic peak aI followed a square root dependence, indicating its diffusional control, up to scan rates near 1 V/s, and then the peak height increased faster until at 50 V/s the anodic peak shape showed clearly effects of adsorption of Thz derivatives at the mercury electrode surface (Fig. 1, curve 1). The cyclic voltammetric experiments revealed the complex behaviour of Thz derivatives at mercury electrodes. Besides reflections of the chemical transformations of the oxidation products, they exhibited secondary effects due to accumulation of the oxidation products at the electrode surface. Such effects of product accumulation and transformation are least pronounced in normal pulse (NP) voltammetric experiments [19,24], and the latter technique was used to study the initial steps in the reaction of Thz derivatives with mercury ions, particularly in attempts to determine whether mercurous or mercuric ions are involved in these reactions. Undisturbed NP voltammograms could be obtained at concentrations of Thz derivatives, chosen such that the amounts of oxidation products formed during the pulse time were limited to ca. 10% of the monolayer electrode coverage [19]. Under these conditions one well developed anodic NP voltammetric wave was obtained for each Thz derivative studied (Fig. 3). The NP waves for PrThz exhibited diffusion controlled limiting current, as was indicated by their pulse width dependence. The half-wave potential of the PrThz waves was independent of the pulse width and PrThz concentration within wide ranges (pulse widths from 3 to 200 ms, and concentrations from 1 to 500 PM were used). This indicates a reversible oxidation process with little effects of product accumulation and transformation. Also, log{ i/( i, - i)} vs. E plots for the NP waves were linear, although their slopes depended to some extent on the pulse width and PrThz concentration. Most of the waves exhibited slopes between 50 and 60 mV, with the lower values observed for smaller pulse widths and lower PrThz concentrations. These slope variations may result from the effects of adsorption of PrThz molecules on the electrode surface. The effects of reactant adsorption were even more pronounced for AThz, for which, at lower concentrations and/or shorter pulse widths, NP waves with a distinct
134 I
I
’
&
OT
1
0.1
2
0
-O.l_
_
-_I
GJJ a
=3
‘-
0
-0.1
--- I'
0.1 -
3
O-
0 L
-O.l-A I -0.1
-0.2
I -4 -0.3
E/V
Fig. 3. Effect of pulse width on normal pulse voltammograms of 1 x 10m4 M PrThz. t,/ms the curves. Phosphate buffer, pH 7.4.
/ -".5E / v
indicated on
Fig. 4. Changes in the cyclic voltammogram upon storage of 5 x lo-’ M PrThz in phosphate buffer, pH 6.0. Time after solution preparation: (1) 0, (2) 1, (3) 6 h. v = 16 mV/s.
maximum were observed. Such behaviour indicates stronger adsorption of the reactant than of the product of the electrode process [25,26]. The NP waves of Thz and AThz oxidation exhibited a pulse width dependence of their half-wave potentials, which shifted to less negative values at shorter pulse widths. This may be attributed to effects of the follow-up transformations of the oxidation products. In spite of the additional effects of adsorption and follow-up chemical transformations, the NP results indicate interactions of the Thz derivatives with Hg(1) ions as the initial step in the anodic process. Also, the width of the anodic peaks observed in OSW voltammetric experiments, close to 120 mV, indicates a one-electron oxidation process. The follow-up transformations evident in the cyclic voltammetric experiments reflect the instability of the initially formed complexes of Thz derivatives with Hg(1) which transform into mercaptides. This was indicated by the potentials of the peaks $/a,, which appeared in a region characteristic for compounds with sulphydryl groups. In spite of difficulties in discriminating between mercurous cysteinate and compounds of mercury with N-substituted cysteine and/or with the respective Schiff bases, some indications of the initial formation of the latter compounds were observed. Thus, there were differences between the shapes and potentials of the c, peaks for different Thz derivatives, and for AThz the c/a,
135
peak couple appeared at potentials more negative than the peaks of L-cysteine (Fig. 2). Decomposition reactions Thiazolidine ring-opening reactions were observed not only after interactions of Thz and its derivatives with Hg(1) ions. Aqueous solutions of the compounds studied were also unstable in the absence of mercury ions. This was reflected in changes in the results of electrochemical experiments observed after storing solutions of Thz and its derivatives for the prolonged times. The cyclic voltammograms in Fig. 4 show a diminishing of the original anodic peak due to PrThz and the simultaneous appearance and increase of an additional peak at more negative potentials. Six hours after preparing the solution, all PrThz had decomposed and only the more negative anodic peak was observed. The last cyclic voltammogram in Fig. 4 is identical to that obtained in a solution of L-cysteine. Also, OSW voltammograms recorded in a solution of the decomposition products exhibited the same shapes of the forward and reverse currents and the peak potentials as those for an L-cysteine solution (Fig. 5). These results show that during the decomposition of
-0.1
-0.3
-0.5 E/V
-0.5
E/V
-1.0
Fig. 5. OSW voltammograms of the decomposition products of PrThz (1) and L-cysteine (2). Phosphate buffer, pH 6.0. (- - -_) Forward and reverse currents, () net current. Fig. 6. Sampled dc polarograms of L-cyst&e (1) and PrTbz (2) in air-saturated phosphate buffer, pH 6.0. Drop and sampling times 1 and 0.02 s. Time (in min) after solution preparation indicated on the curves.
136
COOH
/-I
HS
0 NH2
+
R&H
Scheme 1.
Thz derivatives in aqueous solutions, L-cysteine is liberated and can be detected and identified by electroanalytical techniques. On the other hand, intermediate products of the decomposition of Thz derivatives (Scheme 1) could not be detected in such experiments. In freshly prepared solutions of Thz and its derivatives only one anodic wave was observed, and no open-ring intermediates could be detected, even in the absence of cysteine. Also, during the decomposition no additional wave appeared. This indicates distinctly lower concentrations of the intermediates than of L-cysteine. At least for AThz the potential difference between the anodic and cathodic peaks due to cysteine and the open-ring intermediates (see above) would have allowed the detection of intermediates at concentrations comparable to that of the amino acid. Simultaneous monitoring of the changes in the concentrations of Thz derivatives and L-cysteine enabled kinetic analysis of the decomposition reaction. Two pulse voltammetric techniques were used in such experiments. OSW voltammetry exhibited the virtues of speed and better identification through the shapes of the forward and reverse currents as well as the peak potentials, while suffering from decreased accuracy of the measurements for overlapping peaks. On the other hand, the additive waves observed in the NP voltammetric experiments enabled more accurate measurements of the changing concentrations of Thz derivatives and L-cysteine. The long-time experiments were tested additionally for effects of the presence of oxygen on the reactions studied. It was found that the decomposition rate of Thz and its derivatives is distinctly higher in the presence of oxygen (see below). The sampled dc polarograms in Fig. 6-1 show the oxidation of cysteine by oxygen. The anodic cysteine wave decreased in time and a cathodic wave, due to cystine reduction, appeared and gradually increased. DC polarograms recorded in PrThz solution in the presence of oxygen (Fig. 6-2) also show the formation of cystine. Since cystine may be formed not only by cysteine oxidation but also in reactions of oxygen with intermediate products of the decomposition of Thz derivatives, the kinetics of the decomposition reaction were studied in carefully deaerated solutions. Examples of typical pulse voltammetric curves obtained in studies of the kinetics of the decomposition of Thz derivatives are shown in Fig. 7. Distinct changes in the
137
1o-3t/s Fig. 7. OSW (1) and NP (2) voltammograms of PrTbz in phosphate buffer, pH 6.0. Time (in min) after solution preparation indicated on the curves. Fig. 8. Kinetic analysis of the decomposition of PrThz at pH 6.0 (1) in the absence and (2) in the presence of oxygen.
pulse voltammetric curves, observed after storing the PrThz solution, reflect the decomposition of the Thz derivatives and the formation of the reaction product L-cysteine. Apparent differences between the relative changes of the heights of the OSW voltammetric peaks and the NP voltammetric waves result from differences in their concentration dependences, i.e. in their sensitivities for Thz derivatives and cysteine. The net current in OSW voltammetry, the difference between the forward and reverse currents, is larger than either of the contributions for reversible systems, such as cysteine/cystine (Fig. 5). On the other hand, the chemical irreversibility of the oxidation of Thz derivatives (see above) causes a significant decrease of the OSW voltammetric net current, which, in such cases, is smaller than the forward current. Diffusion controlled limiting currents in NP voltammetry are independent of follow-up chemical transformations and the slight differences between the sensitivities observed for Thz derivatives and cysteine result solely from differences between the respective diffusion coefficients. In order to control the decomposition reactions better, the kinetic analysis was performed in terms of time dependences of the concentrations, rather than of the directly measured electroanalytical signals. Using linear calibration plots determined for Thz and its derivatives and for cysteine at each pH studied (small but distinct pH dependences of the respective sensitivities were observed for cysteine as well as Thz derivatives), the concentration vs. time dependences were analysed. Throughout the decomposition reaction, the sum of the concentrations of Thz derivative and L-cysteine was equal to the initial concentration of the former compound. The latter relation was used as an additional
138
test of the accuracy of the measurements of the varying concentrations and yielded relative standard deviations below 5%. The variations of the concentrations of PrThz during the decomposition reactions followed pseudo-first order kinetics. Linear regression for the dependence In c = k,t + In c0 for PrThz at pHs from 4.5 to 7.4 yielded correlation coefficients better than 0.99 (Fig. 8, curve 1). The slopes of this dependence afforded values of the apparent decomposition rate constant (k,). The k, values obtained for PrThz decomposition from results of NP and OSW voltammetric experiments agreed within 5% and the relative standard deviations of the individual linear regression were below 10%. Determinations of the decomposition rate constants for Thz and AThz were less accurate. This was caused by their much slower decomposition, resulting in small changes in the concentration of Thz derivatives. During an average 8 h long experiment, the decrease in Thz or AThz concentration amounted to only ca. 10% of the initial concentration. An additional difficulty impeding studies of AThz decomposition was caused by a small difference between the half-wave and peak potentials for AThz and L-cysteine (Fig. 2). Therefore, the relative standard deviations for k, determinations under these conditions reached 30%. The adherence of the decomposition reaction to a first-order rate law was tested additionally in experiments performed at different initial concentration of PrThz, from 1 x 10e5 to 1 X 1O-3 M. Experiments at higher concentrations of PrThz were performed in a different way to check the decomposition reaction for a possible dependence on the presence of small amounts of metallic mercury, which was difficult to avoid in the usual pulse voltammetric experiments. In these experiments the decomposition reaction proceeded outside the electrochemical cell and samples of the reaction mixture were analysed immediately. The decomposition rate constant was found to be independent of the presence of small amounts of mercury and of the initial PrThz concentration, except for its highest value. The k, value obtained at an initial concentration of PrThz equal to 1 X 10e3 M was almost a factor of two lower than at low concentrations of the Thz derivatives. This may indicate a change in the decomposition kinetics at high concentrations of Thz derivative, which are, however, of less biological significance. The mean values of the decomposition rate constants of Thz derivatives are collected in Table 1 and show the distinct dependence of the decomposition kinetics on pH and on the nature of the substituent at the C-2 position of Thz. In addition to kinetic measurements of the decomposition of Thz derivatives in carefully deaerated solutions, monitoring of the decomposition reactions in the presence of oxygen was performed in sampled dc polarographic experiments. A change in the mechanism and kinetics of the decomposition reaction was observed: a distinct cathodic wave reflected the formation of cystine (Fig. 6, curve 2). In spite of a different decomposition mechanism, however, the time dependence of the decreasing concentration of PrThz at pH 6.0 followed a pseudo-first-order rate law with correlation coefficient 0.993 (Fig. 8, curve 2). The apparent decomposition rate constant in the presence of oxygen was nearly a factor of two larger than in the deaerated solution. The slower decomposition reactions of PrThz at pH 7.4 and of Thz at pH 6.0 exhibited more complex time dependences. Only during the initial 3 h
139
TABLE 1 First-order rate constants for the decomposition of Thz and its derivatives Compound
PH
Thz
7.4 6.0 6.0+4
10’ k,/s
a
0.06 0.3 1.3
PrTbz
8.0 7.4 7.4+0* a 6.7 6.0 6.0+4 ’ 5.2 4.5
0.03 1.6 2.3 4.3 7.3 14.8 4.7 2.1
ATlIZ
7.4 6.0
0.08 0.3
a Air-saturated solution.
were In c vs. t plots linear, yielding k, values distinctly higher than in the absence of oxygen (Table 1). In experiments on longer time scales an additional acceleration of the decomposition reactions was observed. DISCUSSION
Electrochemical studies of the reactions of Thz derivatives in aqueous solutions showed the importance of the ring-opening reaction in the chemistry of these compounds. This reaction follows the initial formation of unstable complexes of Thz derivatives with Hg(1). It is also the initial step in the decomposition reaction of Thz derivatives in aqueous solutions. Fast scan cyclic voltammetric and pulse voltammetric experiments revealed the initial steps in the anodic processes of Thz and its derivatives at mercury electrodes. The formation of unstable complexes of Thz derivatives with Hg(1) ions is followed by the opening reaction of the thiazolidine ring in which mercaptides are formed. Further transformations of the mercury compounds lead to the formation of cysteine. These findings differ from the assumptions of Costa Garcia and Tunon Blanco [18], who, using slow scan cyclic voltammetric and cathodic stripping experiments, did not observe the initial oxidation products. Therefore, they assumed that, in the anodic reaction of Thz at the mercury electrode, L-cysteine is formed and reacts with mercury ions. Electroanalytical studies of aqueous solutions of Thz and its derivatives showed their decomposition, in which L-cysteine is liberated. No open-ring intermediates of the decomposition were detected either in freshly prepared solutions of the Thz derivatives studied or during their decomposition. The detection limits of the pulse voltammetric methods used would have allowed observation of compounds with sulphydryl groups in concentrations as low as 1 PM. Thus, the results indicate
140
rather low equilibrium concentrations of the respective Schiff bases, which were not observed even in freshly prepared solutions containing no cysteine. The proximity of the anodic waves of various compounds with sulphydryl groups hindered measurements of the open-ring decomposition intermediates (Scheme 1) in distinctly lower concentrations. Still, a detection of these compounds in concentrations comparable to that of cysteine should be possible, at least for AThz. For Thz and PrThz, the structural differences between the open-ring intermediates and L-cysteine are much smaller and the transformations of the mercaptides are probably faster than for the sugar derivative. Therefore it is difficult to judge which of these two reasons precluded detection of the intermediate mercaptides. Because of these difficulties, it is impossible to estimate the positions of the anodic signals due to the open-ring intermediates of the decomposition of Thz and PrThz. However, it seems probable that not only for AThz, but also for the other Thz derivatives the decomposition intermediates appear in low concentrations. This indicates the relative instability of the intermediates and suggest that the thiazolidine ring-opening reaction is the rate determining step in the decomposition of Thz and its derivatives studied. Kinetic measurements of the rate of decomposition of Thz and its derivatives in aqueous solutions showed the distinct dependence of the decomposition rate constant on the nature of the C-2 substituent and on pH. These findings may aid in understanding the biological functions of the compounds studied. Formation and decomposition reactions of thioproline derivatives have been reported to be involved in several biochemical cycles and their function as latent L-cysteine, liberating this important amino acid intracellularly, has often been assumed. In this perspective the observed significant dependence of the decomposition rate of Thz derivatives around physiological pH is particularly interesting. It indicates that non-enzymatic liberation of L-cysteine in vivo may be induced by changes in pH. A pH dependence of the decomposition of 2,2-dialkyl- [15] and 2,2,5,5_tetramethyl- [17] Thz has been observed in previous studies. There is a discrepancy, however, between these reports concerning pHs above 7. Pesek and Frost [17] reported an increase in the decomposition rate at pHs higher than 7, while Riemschneider and Hoyer [15] observed a distinctly slower decomposition at pH 8.8 than at pH 6.8. These investigations of particularly unstable Thz derivatives, performed at high concentrations and apparently in the presence of oxygen, are difficult to compare with the present work, concentrated on studies of Thz and its 2-substituted derivatives representing two groups of prodrugs of L-cysteine used by Nagasawa and co-workers [12-141, and performed at low concentrations and in the absence of oxygen. The effect of the nature of the C-2 substituent on the rate of decomposition correlates clearly with the relative efficacies of Thz derivatives in their protection against the hepatoxic action of acetaminophen [12-141. PrThz has been reported to be much more liver-protective than Thz, which exhibited an efficacy similar to that of AThz. This correlation supports the hypothesis of Nagasawa and co-workers [12-141, who assumed that the intracellular non-enzymatic liberation of L-cysteine causes the hepatoxicity protection by supplying this amino acid precursor of glutathione to the cell.
141 ACKNOWLEDGEMENTS
We thank Jan Radomski for preparation of the Thz derivatives. This work was supported financially by the CPBP.Ol.15 research program. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
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