Enzymatic Iodination of Maleic and Fumaric Acids Diethyl Esters

MARIUS TUDORASCU1*, SPIRIDON OPREA2, AFRODITA DOINA MARCULESCU3, STEFANIA TUDORASCU4 1Office for Pedological and Agrochemical Studies, 131 Lanternei Str., 023093, Bucharest, Romania 2Gh. Asachi University, 3 Bahluiului Str., 700029, Iasi, Romania 3 University of Medicine and Pharmacy, Gr. T. Popa, 16 Universitatii Str., 700115, Iasi, Romania 4 Institute of Physical Chemistry, 202 Splaiul Independentei, 060022, Bucharest, Romania

Maleic and fumaric acids as unsaturated dicarboxylic acids cannot serve with good results for enzymatic transformation [1,2].Maleic acid has a strong acid character [3] and fumaric acid has a low solubility in water, i.e. 0.7% at 25 o C [4], but they can be easily transformed into derivatives such as ionic salts and esters from which, the most important derivatives are diethylmaleate and diethylfumarate.
Our earlier studies showed that the catalytic iodination activity of lactoperoxidase with respect to maleic and fumaric acids and diethyl esters [1,2] in disperse system as substrates took place in the presence of an ionic halide (in this case, iodides) and in the presence of hydrogen peroxide in diluted solution; but with very good results in the presence of a hydrogen peroxide-generating source and in our case in the presence of a system including ethanol and immobilized alcohol oxidase.
Thus, by working with diluted hydrogen peroxide it must be preferably to use very low concentration of 0.5-1% [5] in order to prevent the enzyme inhibition.The halogen source may be any of the water (and alcohol) soluble iodide salts; the preferred halogen sources for the lactoperoxidase-enzyme are calcium and ammonium iodides.These ionic salts must be also used in diluted solutions.
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 in the desired range by using a buffering agent.Suitable buffers included sodium or calcium phosphates, sodium or calcium citrates, sodium or calcium formates, etc [10,11].
Other enzymes such as chloroperoxidase, myeloperoxidase, bromoperoxidase and horseradishperoxidase have a low catalytic effectiveness with respect to diethylmaleate/fumarate iodination.In conclusion, the lactoperoxidase especially in immobilized form as biological catalyst is considered as the best catalyst for this process.

Materials and methods
Diethylmaleate, diethylfumarate, lithium hydroxide, potassium hydroxide, ammonium iodide and calcium iodide, respectively, were provided from Fluka Co; * Tel.: 0723 147 924 lactoperoxidase and alcohol oxidase were provided from Sigma/Aldrich Co, while hydrogen peroxide and ethanol were purchased from CHIMOPAR S.A Bucharest.The inorganic immobilizing agent, such as SAPO-34 was provided from ZECASIN S.A, a research institute of Bucharest.
Thus, both lactoperoxidase and alcoholoxidase were immobilized on SAPO-34, an inorganic silicoaluminophosphate support.The enzyme uptakes were about of 5 units lactoperoxidase activity per gram of support and 10 units alcoholoxidase activity per gram of support, respectively.One unit lactoperoxidase activity forms 1.0 mg of purpurogallin from pyrogallol in 20 sec at pH 6.5 and 20 o C since one unit of alcohol oxidase activity oxidizes 1.0 ìmole of methanol to formaldehyde/minute at pH 7 and 20 o C. For the case of working with free hydrogen peroxide, the reaction system contained substrate: anionic iodide: hydrogen peroxide in a molar ratio of 1: 1: 1.For the case of working with in situ generated hydrogen peroxide, the reaction system contained substrate: anionic iodide in a molar ratio of 1: 1 at which the attaching system contained 300 mmoles of ethanol and 300 units of alcohol oxidase.
In both processes, before enzymatic iodination reaction the two immiscible phases such as organic phase containing organic substrate and aqueous phase containing hydrogen peroxide and ionic iodides in diluted solutions were transformed in disperse system after an ultrasonic pretreatment.Thus, the obtained disperse system was subjected to iodination reaction with immobilized lactoperoxidase in a mixture with immobilized alcoholoxidase, respectively in a column-type reactor.
After one hour of reaction at 20 o C the soluble product such as diethyl-2, 3-diiodosuccinate in a mixture with non reacted diethylic maleic/fumaric substrates were analyzed on atomic absorption spectrophotometer as monoethylpotassium salt (very low soluble in water) for potassium content evaluation.

Results and discussion
Diethylmaleate/fumarate as organic substrates were subjected to the catalytic iodination on immobilized lactoperoxidase in the 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) should be completed with: This serves as iodination agent for equations ( 1) and ( 2), respectively.Observation: LP -immobilized lactoperoxidase; AO -immobilized alcohol oxidase.All described reactions take place at room temperature in a pH range below 7, which is necessary for the stability of enzyme/enzymes.Thus, in accordance with equations ( 1) and ( 2), the enzymatic iodination reaction product consists in diethyl -2, 3diiodosuccinate as a sole product.This iodination compound is spectrophotometrically analyzed as very low soluble 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, 3-diiodosuccinate all water soluble with great errors for potassium content evaluation.
The potassium content after the reaction (4) was about 8.86% in accordance with the chemical composition of this product.
The enzymatic reaction product was then hydrolyzed to tartaric acid presented in an inactive form.Thus, the oxidized product was determined by chemical means (with ammonium vanadate) [12].
All the enzymatic iodination reactions were monitorized in the two phases and disperse system, respectively.The obtained amounts and conversion in 2, 3-diiodide product after enzymatic transformation process are presented in table 1 (after working with free H 2 O 2 in diluted solutions) and table 2, respectively (after working with H 2 O 2generating system), the halide source in both cases being ammonium and calcium iodides.
From both table 1 and table 2 one can see that the iodinated products conversions have much higher values in the case of disperse working system in comparison with those in two-phase system.Also, from both tables one can observe that diethylmaleate is a more active substrate than diethylfumarate, calcium iodide is a more active halide source than ammonium iodide and working in disperse system better results are obtained with respect to conversion values especially when hydrogen peroxide is in situ generated.Thus, the conversion into diethyl-2, 3-diiodosuccinate variation versus time for each halide source is presented in figures 1a and 1b, for the case with free H 2 O 2 working and in figures 2a and 2b, respectively, for the case with in situ H 2 O 2 -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 figure 3 is presented the lactoperoxidase-enzyme activity variation during the diethylmaleate/diethylfumarate iodination process versus the pH values of the reaction in disperse system in which the pH values below 6.7 (considered as favorable value for the maximal activity of this enzyme) were adjusted by addition of very small amounts of diluted HI solution; while the pH values raised over 6.7, the pH was adjusted by addition of very small amounts of diluted ammonia solution.
Our discovery that lactoperoxidase catalyses iodination reaction of diethylmaleate/diethylfumarate in disperse system to iodination product such as diethyl-2, 3diiodosuccinate as sole product is quite interesting in view of current knowledge concerning the enzymatic processes in organic chemistry.These reaction types constitute a great help to elucidate the mechanism (long time unknown) in which the enzyme is implicated.Thus, during this enzymatic process the iodinated product is present only as diethyl-2, 3-diiodosuccinate without diethyl-αiodomalate.The presence of the diiodinated compound as sole product according with the following mechanism: (5) The final diiodinated product such as diethyl-2, 3diiodosuccinate is easily hydrolyzed (at 80 o C) with the forming of inactive optical form of tartaric acid and ethanol, both starting with diethylmaleate and diethylfumarate as substrates.For the case of diethylmaleate as substrate, the final hydrolyzed product was D, L-tartaric acid, whereas for the case of diethylfumarate as substrate the final hydrolyzed product was meso-tartaric acid.Why? a) For the case of diethylmaleate, according to the following equations due to the fact that there are polar groups the halide takes place following the ordinary pathway without rotating the carboxyl group with 180 o resulting the iodinated optically active products in a racemic mixture.Thus, the final reaction is: b) For the case of diethylfumarate, according to the following equations due to the fact that there are no polar groups the halide takes places in an unordinary pathway by rotating with 180 o of the carboxyl group to the molecule plane after appearance of a cyclic halonium (iodonium) cation as intermediate (11).
In conclusion, after final hydrolysis of resulted iodinated compound starting from diethylmaleate (after enzymatic iodination in disperse system), D, L-tartaric acid results, whereas after final hydrolysis of iodinated compound starting from diethylfumarate (after enzymatic iodination in disperse system), meso-tartaric acid 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 bitartrate as initial ionic product results.If tartaric acid is in its racemic form, from the aqueous D, L-lithium bitartrate after splitting in few hours the two enantiomers result, such as L (+)-lithium bitartrate in precipitate form with a low solubility, whereas D (-)lithium bitartrate (the second enantiomer) remains in aqueous solution, which is soluble.But, in the case of meso-tartaric acid in reaction with lithium hydroxide in a same molar ratio one can observe that the resulted soluble meso-lithium bitartrate exists permanently in aqueous solution without splitting into enantiomers.

Conclusions
The obtained results in a disperse system are much better comparatively to those obtained in a two-phase system.Utilization of the disperse system in the process of diethylmaleate and diethylfumarate iodination in the presence of immobilized lactoperoxidase as enzyme, leads to a new reaction mechanism supported by the racemic and meso-final conformation.
The obtained results using immobilized lactoperoxidase during the iodination process in disperse system leads us to new opportunities in food, agriculture and pharmaceutical industry development and for example, for choline, acetylcholine and butyrylthiocholine bitartrates synthesis.

( 3 )
Fig.1b.The time dependence of conversion into diethyl-2, 3diiodosuccinate in disperse system during enzymatic iodination process working with free H 2 O 2 .Substrate diethylfumarate; halide source NH 4 I