ENZYMIC IODINATION OF MALEIC AND FUMARIC ACIDS DIETHYL ESTERS

ENZYMIC IODINATION OF MALEIC AND FUMARIC ACIDS DIETHYL ESTERS

SUMMARY

The mechanism of the enzymic iodination process of diethylmaleate and diethylfumarate in the presence of lactoperoxidase, both in diluted hydrogen peroxide solution and in a generating-system of hydrogen peroxide, using ammonium and calcium iodides as halide sources, in disperse system was studied.

The obtained sale product (diethyl-2 -diiodosuccinate) after the enzymic iodination process was directly hydrolyzed to a tartaric acid, presented in an optically inactive form. The mechanism of obtaining the intermediate and final products and respectively, the existence of both, D,L-tarteic acid and meso-tartric acid (as lithium bitartrates) were also investigated.

Key words: lactoperoxidase, disperse system, ammonium/calcium iodides, lithium bitartrates.

INTRODUCTION

Maleic and fumaric acids as unsaturated dicarboxylic acids cannot serve with good results as substrates for enzymic transformation |1, 2|; maleic acid as a strong acid character |3| and fumaric acid owing to a low solubility in water (0,7% at 25^oC |4|), but thei can be easily transformed into derivates, such as: ionic salts and esters, the most important derivatives being diethylmaleate and diethylfumarate.

Our earlier studies showed that the catalytic iodination activity of lactoperoxidase with respect of maleic and fumaric acids, diethylesters |1, 2| in disperse system as substrates take place in presence of an ionic halice (in this case, iodides) and in presence of hydrogen peroxide in diluted solution; but with very good results, in presence of an hydrogen peroxide-generating source, in our case, in presence of a system including ethanol and immobilized alcohol oxidase.

Thus, by working with diluted hydrogen peroxide, it must be preferably used in very low concentration (0,5-1% |5|) necessary to prevent the enzyme inhibition. The halogen source may be any of the water (and alcohol) soluble iodide salts; the preferred halogen source for the lactoperoxidase-enzyme are calcium and ammonium iodides. These ionic salts must be also used in diluted solution.

The iodination reactions catalyzed by lactoperoxidase are conducted within the pH range of from 6 to about 7. In many cases, the pH-value of the reaction may be maintained within the desired range by use a buffering agent. Suitable buffers included: sodium or calcium phosphates, sodium or calcium citrates, sodium or calcium formates etc. |10, 11|.

EXPERIMENTAL METHODS

The enzymes immobilization was carried out in agreement with Neidleman & coworkers processing [5 9].

Thus, both lactoperoxidase and alcoholoxidase were immobilized on SAPO-34, an inorganic phospho-zeolyte support. The enzymes uptakes were about: 5 units lactoperoxidase activity per g support and respectively 10 units alcoholoxidase activity per g support. One unit lactoperoxidase activity forms 1,0 mg purpurogallin from pyrogallol in 20 sec. at pH 6,5 at 20^oC, since one unit alcoholoxidase activity oxidize 1,0 mol of methanol to formaldehyde per min. at pH 7, at 20^oC.

For the case with free hydrogen peroxide working, the reaction system contained: substrate : anionic iodides : hydrogen peroxide, in molecular ratio of 1:1:1. For the case with in situ generated hydrogen peroxide working, the reaction system contained substrate : anionic iodides, in a molar ratio of 1:1, at which the attaching system contained 300 mmols ethanol and 300 units alcohol oxidase.

In both process, before enzymic iodination reaction, the two immiscible phases such as: organic phase contained organic substrate and aqueous phase contained hydrogen peroxide and ionic iodides in diluted solutions were transforming in disperse an ultrasonic pretreatment.

Thus, the obtained disperse system was subjected to iodination reaction with immobilized lactoperoxidase in mixture with immobilized alcohol oxidase respectively, in a column-type reactor.

After 1 hour of reaction at 20^oC, the soluble product such as: diethyl-2 -diiodoscuccinate in mixture with unreacted diethylic maleic/fumaric substrates, were analyzed on atomic absorption spectrophotometer, as monoethyl potassium salt (very low soluble in water) for potassium content evaluation.

RESULTS DISCUSSION

Diethylmaleate/fumarate as organic substrates were subjected to the catalytic iodination, on immobilized lactoperoxidase in presence of ionic iodides both in diluted hydrogen peroxide and in hydrogen peroxide in situ generation system, such as:

When, we work with hydrogen peroxide in situ generation, the equations (1) or (2) were completed with:

Which serves as iodination agent for the eqs. (1) and (2) respectively reactions.

Thus, conforming with the eqs. (1); (2), the reaction product during the enzymic iodination process consists in diethyl-2 -diiodosuccinate, as a sole product. This iodination compound is analiyed by spectrophotometrically mean, as monoethylpotassium salt, after a preliminary reaction:

for potassium content evaluation.

The reaction (4) takes place at room temperature, to prevent other organic products forming, such as: dipotassium epoxysuccinate or dipotassium oxalylacetate and especially dipotassium 2 -diiodosuccinate, all soluble in water, with great errors for potassium content determination.

The potassium content after the reaction (4) was about 8 % in agreement with the chemical composition of this product.

The enzymic reaction product was than hydrolyzed to tartaric acid presented in inactive form. Thus, this oxidized product was determined by chemical means (with ammonium vanadate) in accordance with Merck & Co method [12].

All the enzymic iodination reactions were monitorized in two phase and disperse system respectively. The obtained amounts and conversion is 2,3-diiodide product after enzymic transformation process, are presented in Table I (after working with free H₂O₂ in diluted solution) and Table II (after working with a H₂O₂ generating system), the halide source in both tables, being ammonium and calcium iodides.

TABLE I. Amounts (Q) and conversion (h) of the enzymic reaction product (diethyl-2 -diiodosuccinate) for the catalytic immobilized lactoperoxidase reaction. Conditions: substrates: diethylmaleate/fumarate (20 mmols); halides: NH₄I (20 mmols) and CaI₂ (10 mmols); H₂O₂ in initial form; system: two-phase and disperse; biocatalyst: lactoperoxidase (LP) immobilized on SAPO-34, inorganic support (200 units activity); reaction time: 1 hour; temperature: 20^oC.

TABLE II Amounts (Q) and conversion (h) of the enzymic reaction product (diethyl-2 -diiodosuccinate) for the catalytic immobilized lactoperoxidase reaction. Conditions: substrates: diethylmaleate/fumarate (20 mmols); halides: NH₄I (20 mmols) and CaI₂ (10 mmols); H₂O₂ in situ generated by the system: ethanol (300 mmols) immobilized alcohol oxidase (AO) (300 units); system: two-phase and disperse; biocatalyst: lactoperoxidase (LP) immobilized on SAPO-34, inorganic support (200 units activity); reaction time: 1 hour; temperature: 20^oC.

From both table I and Table II one can see that in the case of dispers system, the iodinated products conversions have much higher values in comparison with those in two-phase system. Also, from both tables, one can observe that: diethylmaleate is more active substrate than diethylfumarate, calcium iodide is more active halide source than ammonium iodide, and the working in disperse system gives better results with respect of conversion values, especially when hydrogen peroxide is in situ generated.

Thus, the conversion into diethyl-2 -diiodosuccinate variation versus Time for each halide source in disperse system after enzymic iodination process is presented in fig. F-1, for the case with free H₂O₂ working and in fig. F-2 respectively, for the case with in situ H₂O₂ generating system working.

The synthesis of iodination products depends upon the lactoperoxidase enzyme activity, which depends in its turn upon the pH values of the reaction system. Thus, in fig. F-3 is presented the lactoperoxidase-enzyme activity during the diethylmaleate/fumarate iodination process, versus the pH values of the reaction in disperse system, in which the pH values below 6 (considered as a value in concordance with the maximal activity of this enzyme) were adjusted after addition of very small amounts of diluted HI solutions; while the pH values over 6,7 were adjusted after addition of very small amounts of diluted NH₃ solution.

Our discovery that lactoperoxidase catalyses the iodination reaction of diethylmaleate/fumarate in disperse system to iodination product such as: diethyl-2 -diiodosuccinate as sole product are quite interesting in view of current knowledge concerning the enzymic process in organic chemistry. These reactions types constitute a great helpfulness for the owing to elucidate the mechanism (more time unknown) in which the enzyme is implicate. Thus, during this enzymic process, the iodinated product is presented only as diethyl-2 -diiodosuccinate, without diethyl-a-iodomaleate. The presence of the diiodinated compound as sole product is in concordance with the fallowing mechanism:

  (5)

  (6)

very unstable

  (7)

Thus, the final reaction is:

The final diiodinated product, such as: diethyl-2 -diiodosuccinate is easily hydrolyzed (at 80^oC) with formic of inactive optical form of tartaric acid, hidroiodis acid and ethanol, both starting with diethylmaleate and with diethylfumarate as substrates. For the case of diethylmaleate as substrate, the final hydrolyzed product was D,L-tartric acid, since for the case of diethylfumarate as substrate, the final hydrolyzed product was mesotartric acid. Why?

a) For the case of diethylmaleate, conforming with the following eqs:

due to the fact, there an polar groups, the halide takes place fallowing the ordinary pathway, without rotating the carboxyl group with 180^o, resulting the iodinated optically active products in racemic mixture.

b) For the case of diethylfumarate, conforming with the following eqs:

  (9)

due to the fact, there are no polar group, the halide takes place in an unordinary pathway, by rotating with 180^o of the carboxyl group to the molecule plane after appearance of a cyclic halonium cation as intermediate (see eq. 11).

In conclusion, after final hydrolysis of resulted iodinated compound starting from diethylmaleate, D,L-tartaric acid results, since after final hydrolysis of iodinated compound starting from diethylfumarate, meso-tartaric results.

The existence of tartaric acid, both in racemic and in meso form, as final hydrolysis product, was pointed out from its reaction with lithium hydroxide in a molar ratio about 1 : 1, when soluble lithium bi tartrate as initial ionic product results. If tartaric acid is in racemic form, from the aqueous D, L lithium bitartrate, after splitting, resulted in few hours the 2 enentiomerides, such as: L (+) lithium bitartrate in a precipitant form, with a low solubility, since, D (-) lithium bitartrate (the second enantiomeride) remains in aqueous solution, which is soluble. But in the case of meso-tartaric acid, in reaction with LiOH in a same molar ratio, one can observe that the resulted, soluble meso-lithium bitartrate exists permanently in aqueous solution, without splitting into enantiomerides.

CONCLUSIONS

The obtained results in a disperse system are much better, comparative to those obtained in a two-phase system. Using the disperse system in the process of diethylmaleate and diethylfumarate iodination, in presence of immobilized lactoperoxidase as enzyme, leads to a new reaction mechanism sustained by the racemic and meso, final conformation.

The obtained results using immobilized lactoperoxidase in disperse system, leads us to new opportunities in food, agriculture and pharmaceutical industry development, and example for choline, acetylcholine and butyrylthiocoline bitartrates synthesis.

REFERINCES

|1| Tudorascu, M.; Popa, I.; Tudorascu, St. Roum. Biotechn. Lett., 2 (2), 170+178 (1997).

|2| Tudorascu, M.; Popa, I.; Tudorascu, St. Prog. Cat., 6 (1), p. 49-55 (1997).

|3| Nenitescu, C.D. Chimie Organica, vol. I, Ed. Didactica si Pedagogica, Bucuresti, p. 782 (1973).

|4| Nenitescu, C.D. Chimie Organica, vol. I, Ed. Didactica si Pedagogica, Bucuresti, p. 784 (1973).

|5| Neidleman, S.L. et al. U.S. Pat. Appl. U.S. 4247641, Cls C12P-17/02 (1981).

|6| Neidleman, S.L. et al. Eur. Pat. Appl. E.P. 7176, Cls C07D-301/00 (1978).

|7| Neidleman, S.L. et al. U.S. Pat. Appl. U.S. 4282324, Cls C12P-3/00 (1989).

|8| Neidleman, S.L. et al. U.S. Pat. Appl. U.S. 4284723, Cls C12P-17/02 (1981).

|9| Geigert, J. et al. U.S. Pat. Appl. U.S. 4587217, Cls C12P-7/34 (1986).

|10| Beneson, A. et al. Ger. Offen D.E. 2731921, Cls C07B-29/00 (1978).

|11| Geigert, J.; Amon, Fr.; dabietos, M. Appl. Environm. Microbiol. 45 (5), p. 1578-1580 (1983).

|12| Merck, E. & al. Rapid Test Handbook, 1-st Ed., p. 264-266 (1987).