Electrochemical synthesis of amino acids by reduction of
phenylhydrazones of the folowing a-ketoacids: pyruvic, a-ketovaleric,
a-ketoglutaric and a-ketooctane acid was studied. The
corresponding a-amino acids were obtained in 44--55~/o yield using
controlled potential electrolysis on mercury pool electrode in 0.5 M
HCl aqueous ethanol solution.
A mechanism rationalizing the reduction products of the phenylhydrazone
of pyruvic acid as well as polarographic and coulometric
data are proposed

I. Tabaković

M. Džepina

M. Trkovnik

English

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CROATICA CHEMICA ACTA CCACAA 49 (3) 497-503 (1977) CCA-1022 YU ISSN 0011-1643 547.46 Original Scientific Paper Synthesis of Amino Acids by Cathodic Cleavage of Phenylhydrazones of a-Keto Acids I. Tabakovic*, M. Trkovnik, and M. Dzepina Faculty of 'i.' echnology, University of Banjaluka, 78000 Banjaluka, Bosnia and Hercegovina, Yugoslavia Received January 24, 1977 Electrochemical synthesis of amino acids by reduction of phenylhydrazones of the folowing a-ketoacids: pyruvic, a-keto-valeric, a-ketoglutaric and a-ketooctane acid was studied. The corresponding a-amino acids were obtained in 44--55~/o yield using controlled potential electrolysis on mercury pool electrode in 0.5 M HCl aqueous ethanol solution. A mechanism rationalizing the reduction products of the phe-nylhydrazone of pyruvic acid as well as polarographic and coulo-metric data are proposed. It has been shown in the literature that a number of important amino acids can be synthetised by reduction of esters of substituted a-nitroacrylic acid 1- 6 . Other methods such as electroreductive coupling of Schiff bases with alkyl halides7 and reductive cleavage of the C-S bond8, have been applied in electrochemical preparations of amino acids. Several authors have used electro-chemical cleavage in the peptide chemistrys,9_ Reductive cleavage of phenylhydrazones of a-keto acids is an important method for the synthesis of a -amino acids, because of the easy availability of those phenylhydrazones by the Japp-Klingemann reaction10. V. V. Feofilakov and coworkers11 have described the reduction of phenylhydrazones of a-keto-acids to the corresponding amino acids by means of zinc dust in 750/o alcohol in the presence of mercuric chloride. · In the present work the electrochemical synthesis of a-amino acids by means of reductive cleavage of the corresponding phenylhydrazones was studied. Utilisation of electroanalytical techniques described herein should contribute to the elucidation of the reaction mechanism. RESULTS AND DISCUSSION Phenylhydrazones of the following a-ketoacids were included in the investigations: pyruvic, a-ketovaleric a-ketoglutaric and a-ketooctane acid. With all of the compounds studied well defined polarographic waves were observed, the total height of which was approximately the same for all of the substances compared. Comparison of the wave height of selected phenylhydrazones obtained in 0,5 M HCl solution with waves of equimolar concentrations of benzaldehyde and benzophenone semicarbazone, for which the number of electrons was * The author for correspondence Phenylhydrazone Ei12• of v SC. S.C.E. Pyruvic acid I -0.691 a-Ketovaleric acid -0.685 a-Ketoglutaric acid -0.666 a-Ketooctanoic acid -0.655 I TABLE I Electroanalitical and Preparative Data Potential id" controlled n-Value µA V vs S.C.E. I I 8.0 4.0 -0.9 8.1 3.9 -0.9 8.5 3.8 -0.9 8.4 3.8 -0.9 • 1 X io-s M concentration of phenylhydrazones of tt-keto acid in 0.5 M HCl aqueous ethanol (50'/o) Amino acid obtained m.p. (lit. m.p)0 oc Alanine 294-296 (295-297) a-Aminovaleric acid 298-300 (305) Glutaminic acid 221-224 (224-5) a-Aminooctanoic acid I 286-270 (270) 0 Taken from: R. G. We as t (53rd edition) "Handbook of Chemistry and Physics«, The Chemi<:al Rubber Co., Ohio (1972). Yield O/o ' 51 44 50 55 >I» '° co ~ 8 > td > ~ 0 ~ H () M >'l ;i,. !:""' ELECTROCHEMICAL SYNTHESIS OF AMINO ACIDS 499 known13 is indicating four-electron processes. The results of coulometry at controlled potential, given in Table I, varied between n = 3.8 and 4.0. Logaritmic analysis of polarograms indicated an irreversible nature of the waves. The ir-reversibility was further confirmed by slow sweep cyclic voltammetry. The linear dependence of limiting currents on (a) the square root of the effective height of the mercury reservoir, and (b) the concentration of the phenyl-hydrazons showed that it is diffusion controlled. Main polarographic and preparative results are shown in Table I. The preparative reduction of phenylhydrazones of a-ketoacids were performed in 0.5 M HCl solution (500/o aqueous ethanol) on the mercury pool electrode in a divided cell using electrolysis at controlled potential (- 0.9 V vs. S.C.E.). The corresponding amino acids were isolated in 44-550/o yield. All obtained amino acids gave correct elemental analysis and showed identical m.ps. and ir-spectra with authentic samples. ' The polarographic behaviour of the phenylhydrazone of pyruvic acid, as well as the products of the preparative reduction, were studied in some details. This compound is reduced in a well-defined wave in the whole pH range examined. Several typical polarograms are shown in the Figure 1. STAR T: -0.5 V d.5-~E. Figure 1. Typical polarograms of the phenylhydrazone of pyruvic acid (1 X 10-s M) Below pH = 2.8 the limiting current is controlled by diffusion and practically pH-independent. Above pH = 2.8 wave decreased with the increase of pH in the form of a steep dissociation curve (Figure 2) with pK' equal to 6.2 (pK' is the pH at which i = id/2). Studies made at pH 6.0 showed that id/Ch1/ 2 values were not constant. This fact confirms a kinetically controlled nature of the reduction. A plot of the logarithm of the limiting current against pH gave a curve with a limiting slope of about-1, indicating the participation of one proton in the potential-determining step14• Below pH = 2.8 the half-wave potential is practi-cally pH-independent, while above this value the half-wave potential is shifted to more negative values with increasing pH of the solution. The current is thus an example of the formation of an electroactive species from electroinactive form, diffusing towards the electrode. In pH range 4.2 to 7.2. the 500 I. TABAKOVIC ET AL. w u (j') vi >' > i/id ~ w 1.00 -1 .2 0 0.75 -1. 1 0.50 -1.0 0.25 -0.9 OL-~-,--,-~,--.---.--,-~.--.-~ 0 2 3 4 5 6 7 8 9 pH Figure 2. Dependence of limiting currents expressed as a fraction of the diffusion current i /!d, (o)n .and half-wave potential, (al on pH for the phenylhydrazone of pyruvic acid . limiting current decreased rapidly with increasing pH. This suggests that the height of the wave reflects the extent to which a reaction of the following type occurs during the drop life ~ + e phenylhydrazone + H+ ~ protonated phenylhydrazone -+ product k . . Using the treatment by Koutecky15 and the polarographic value of pK' = 6.2 at t 1 = 2.6ls the rate constant for proton recombination reaction was computed as k1 = 3 X 105 1 mo1-1 s-1 to deduce that the electrode process is accompanied by an antecedent acid-base reaction. The reduction of phenylhydrazone of pyruvic acid is in general similar to the reduction of many azomethine derivative types described by H. Lund15• The electrode process taking place can be represented by the following scheme: ELECTROCHEMICAL SYNTHESIS OF AMINO ACIDS CflJ--C--COOH II N I 0 Scheme 501 According to the scheme the electroactive form in acidic solution is the protonated phenylhydrazone of pyruvic acid. After two electrons and one proton are accepted by the protonated phenylhydrazone a cleavage of the nitrogen-nitrogen bond occurres giving rise to the a-imino propane acid and aniline, as a good leaving group. a-Imino propane acid may be either further reduced through the transfer of two electrons and two protons leading tb alanine, or may be hydrolised to pyruvic acid being a reducible species at applied potential (- 0.9 V) in acidic media and giving rise to lactic acid. Preparative results and the determination of n-value by coulbmetry at controlled potential (n = 4) are fitting within the proposed scheme. Namely, besides alanine we have !dentified by means of TLC only two additional products, e.g. aniline and lactic acid. These results could explain the dis-crepancy between the chemical yield of alanine (510/o) and n-value determined coulometrically (n = 4). EXPERIMENTAL Polarography Polarograms were recorded on a Polariter P04-polarograph (Radiometer Copen-hagen). The cell used was a modified H-type of 25 ml capacity with a dropping mercury electrode and saturated calomel electrode connected via a salt bridge of 30/0 agar in saturated potassium chloride solution. Temperature was kept constant at 25.0 ± 0.1 °c by means of a thermostat. The characteristic of capillary in H20 open circuit are as follows: t = 2.61 s drop-1, m = 3.10 mg s-1 for h = 60 cm. 1.25 X 10-2 M stock solution of phenylhydrazones of a-keto acids were freshly prepared in 500/o ethanol. The buffer solution used were: pH= 1-3 hydrochloric acid-sodium acetate; pH = 3-8 Mc Ilvain buffers. Buffer solutions and all other solutions were prepared using Reagent Grade chemicals. Gelatin was added as maximum suppresor at a concentration of 0.01°/o. 502 I. TABAKOVIC ET AL. Preparative Electrolysis and Coulometry Electrolysis at controlled potential were carried out by means of a potentiostat (Amel-555-SU). The electricity amount was measured by an electronic integrator when the current dropped to 1-3°/o of its starting value and the number of electrons transferred during each experiment was calculated. Preparative electrolysis was performed in the H-type cell previously described16• The cathode was a mercury pool (40 cm2) and graphite was used as anode. A saturated calomel reference electrode was connected to the cathode compartment through a Luggin capillary. All electrolyses were carried out under nitrogen atmosphere. The hydrazone of a-keto acid (0 .. 5-1.0 g) was added to the cathodic compartment of the cell filled with 250 ml of 0.5 M HCl solution in 500/o aqueous ethanol. The potential was maintained at - 0.9 V vs. S.C.E. with initial currents generally 400--800 mA. After stopping the electrolysis the catholyte was evaporated to the volume of 5-10 ml. The residue. was passed through a column (2 X 20 cm) of cation exchange resin (Dower-50) . After thorough washing of the column with distilled water, the amino acid was taken off the column with aqueous ammonia (2°/1>) . The eluate was evaporated to dryness under vacuum to a small volume (5 ml) . To the water solution ethanol was added and the amino acid was precipitated with ether. Electrochemically obtained amino acids showed correct m .ps. and elemental analysis. Thin Layer Chromatography The mixture obtained after the reduction of the phenylhydrazone of pyruvic acid was taken from the catholyte and subjected to TLC analysis. Thin layer chromato-graphy was carried out on HF-silicagel plates (250 µm) dried 30 minutes at 110 °c. The developer used was: n-butanole : acetic acid : water (7 : 2: 1). The 0.4-0/o nynhidrine solution in acetone was used for detection of alanine (Rf = 0.23) and aniline (Rf = = 0.86). The detection of lactic acid (Rf = 0.30) was done by means of 0,20/o solution of bromophenol blue reagent. The Rf values of the mixture components were also compared with authentic samples. REFERENCES 1. E. V. Zaporozhets, I. A. Avrutskaya, M. Ya. Fioshin, K. K. Ba-b i e vs k ii, and V. M. B e 1 i k o v, Elektrokhimiya, 8 (1972) 1243. 2. E. V. Zap or oz he ts, I. A. Av ruts k a ya, M. Ya. Fi o shin, K. K. Ba-b i e vs k ii, and V. M. B e 1 i k o v, Elektrokhimiya, 9 (1973) 270. 3. E. V. Zap o r oz he ts, I. V. Av ruts k a ya, M. Ya. Fi o s hin, K. K. B a-b i e vs k ii, and V. M. B e 1 i k o v, Elektrokhimiya, 8 (1972) 1809. 4. E. V. Zap or oz he ts. I. A. Av ruts k a ya; M. Ya. Fi o shin, K . K . Ba-b i e vs k ii, and V. M. Be 1 i k o v, Elektrokhimiya, 9 (1973) 72. 5. I. A. Av ruts k a ya, M. Ya. Fi o shin, K. K. Bab i e vs k i, V. M. Be-11 i k o v, and E. V. Zap or oz he ts, Elektrokhimiya, 9 (1973) 1127. 6. I. A. Av ruts k a ya, K. K. B ab i e vs k ii, V. M. B e 1 i k o v, E. V. Z a po-r oz he ts, I. M. Fi 1 at o v a, and M. Ya. Fi o shin, Elektrokhimiya, 9 (1973) 1652. . 7. T. Iwasaki and K. Har ad a, Chem. Comm. (1974) 338. 8. T. I w as a k i, M. Miya sh i, M. Matsuo k a, and K. Matsumoto, Chem. Ind. (1973) 1163 and references therein. · 9. L. Horner and H. Lund in : M. M. B a i z er (Ed.). »Organic Electrochemi-stry«, Marcel Dekker, New York, 1973, pp. 729. 10. R. F. Japp and F. K 1 in gem an n, Ber. 20 1 (1887) 2942, 3284, 3398. 11. V. V. Feofilaktov and T. N. Ivanova, Zh. Obsch. Khim. 21 (1951) 1684 and references therein. 12. N. H . Khan and A. R. Kid w a i, J . Org. Chem. 38 (1973) 822. ELECTROCHEMICAL SYNTHESIS OF AMINO ACIDS 503 13. B. F 1 e et and P. Zuma n, Collect. Czech. Chem. Commun., 32 (1967) 2066. 14. R. I. Ge 1 b and L. Mei t es, J . Phys. Chem., 68 (1964) 2599. 15. H. Lund, Acta Chem. Scand., 13 (1959) 249; H. Lund and E. Th. Jensen, Acta Chem. Scand., 25 (1971) 2727 and references therein. 16. H. Lund and P . Iversen in: M. M. Baizer (Ed.) »Organic Electroche-mistry«, Marcel Dekker, New York, 1973. pp. 177. SAZETAK Sinteza aminokiselina katodnim cijepanjem fenilhidrazona a-ketokiselina I. Tabakovic, M. Trkovnik i M. Ditepina Izucavana je elektrokemijska sinteza aminokiselina redukcijom slijedecih fenil-hidrazona a-ketokiselina: pirogrozdane, a-ketovalerijanske, a-ketoglutarne i a-keto-oktan kiseline. Elektrolizom kod kontroliranog potencijala zivine katode u 0,5 M vode-noetanolnoj otopini HCl dobivene su odgovarajuce a-aminokiseline u 44-550/o-tnom iskoristenju. Pretpostavljen je reakcioni mehanizam kojim se objai§njava nastajanje produkata redukcije fenilhidrazona pirogrozdane kiseline, a takoder su dati rezultati polarografije i kulometrije kod kontroliranog potencijala. TEHNOLOSKIFAKULTET UNIVERZITET U BANJALUCI Prispjelo 24. sijecnja 1977. 78000 BANJALUKA, BOSNA I HERCEGOVINA 

Synthesis of Amino Acids by Cathodic Cleavage of Phenylhydrazones of a-Keto Acids

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