The Oxidation of Quinic Acid

The Oxidation of Quinic Acid A. C. HULME AND W. ARTHINGTON Ditton Laboratory, Department of Scientific and Industrial Research Received 26 June 1952 SUMMARY 1. It is shown by means of filter-paper chromatograms prepared at intervals during the oxidation of quinic acid by hydrogen peroxide that at least six acids appear in the reaction liquid. 2. One of these acids is shown to be citric acid, and the oxidation of citric acid is shown to account for a further two of the acids resulting from the oxidation of quinic acid. 3. After prolonged oxidation (by H 2 O 2 ) of both quinic and citric acids one acid predominates. This acid is proved by isolation and characterization to be malonic acid. 4. Evidence is produced which suggests that acetonedicarboxylic acid is an intermediate in the oxidation of citric acid (and, therefore, of quinic acid) to malonic acid. IN the course of the isolation and identification of quinic acid from the young fruits of the Worcester Pearmain apple, filter-paper chromatograms were run at intervals during the oxidation of the quinic acid with hot H 2 O 2 (Hulme, 1951). These chromatograms showed that, while the reaction as a whole is a complex one involving at least six acids in addition to the original quinic acid, two acids emerged as the main products of the reaction. One of these was found to be citric acid, which was the acid obtained by Fischer and Dangschat (1934) on oxidation of quinic acid by periodic acid followed by bromine water. The work described in the present paper was carried out primarily to identify the second acid. The results also illustrate the value of paper chromatography in following the course of a complex series of reactions. The large amount of quinic acid present in young apples recent work has shown that young Worcester Pearmain apples (average weight 15 g.) contain rather more quinic than malic acid poses the question of its function in the metabolism of the fruit as indeed in other plant tissues where it is known to exist. For this reason a more complete knowledge of its chemistry is desirable than at present exists. Small-scale oxidations (~ 20 mg. quinic acid and 1 ml. 100 vol. H 2 O 2 ) yield, as oxidation proceeds, chromatograms having the patterns shown in Fig. 1. Fig. 2 (upper portion) is a photograph of a chromatogram taken after 25-30 hours' oxidation in the large-scale preparative experiment to be described later; it is included to show the streaking effect produced by the continuous state of flux of the system. Although it does not follow that the acid-reacting spots on the chromatogram represent a sequence of events commencing at the acid of lowest R F during Journ. of Experimental Botany, Vol. 4, No. 11, pp. 129-33, June 1953. 5I6O-II

130 Hulme and Arthington The Oxidation of Quinic Acid the course of a reaction an acid of high R F may -be formed before one of low R F oxidation of citric acid by hot H 2 O 2 yields chromatograms which reproduce the second stage (citric acid and acids of higher R F values) of the chromatograms of Figs. 1-2. It will be convenient, therefore to consider the oxidation of quinic acid in two stages, quinic to citric acid and citric to 'X' acid (R F ^ 0-62). H202 + X PRE-HEAT 40MIN.HEAT 80-120 - HC A B <=> o C "> E CD <=> CD O 6 > CD CD CD E o SOLVEN 10cm. FIG. 1. Chromatogram, run in 'B.F.W.', of the_reaction liquid of the oxidation of quinic acid ('X' in the figure) by H 2 O 2 (see text). HC represents a marker-spot of citric acid. EXPERIMENTAL Reagents used. Quinic acid: Purified samples obtained from Worcester Pearmain apples (Hulme, 1951) or commercial samples (Light and Co.; not recrystallized) were used; both gave identical results. Hydrogen peroxide: B.D.H., 100 vol. M.A.R. grade was used. Oxidations were carried out in round-bottom flasks fitted with a 'cold finger', in an oil-bath. Chromatographic methods. Whatman No. 2 paper was used throughout. Down-flowing chromatograms were run in a room kept at a constant temperature of 20 0 C. All papers were equilibrated for 24 hours with the more aqueous layer (where immiscible solvents were used) before addition of solvent. Solvents. The two solvent systems used for the detection of acids were butanol-formic acid-water (40:10:50 v/v) due to Lugg and Overell (1948), hereafter designated 'B.F.W.'; ethanol-ammonia-water (80:4:16 v/v) advocated by Long et al. (1951) ('E.Am.W.'). The papers were dried in a current of air in an oven at no C. Sprays. With B.F.W., bromo-phenol-blue (5 per cent, in 50 per cent, alcohol, made just alkaline with sodium hydroxide) was used to reveal the position of acids. B.D.H. Universal indicator made to ph 9-5 with soda was found to be considerably more sensitive, revealing the presence of much smaller amounts of acid (Long, Quale, and Stedman, 1951). This spray could, however, only be used with the solvent system E.Am.W. since, even after prolonged drying, papers run in B.F.W. remained sufficiently acid to render the whole of the paper sprayed with the alkaline Universal indicator red almost immediately after spraying.

FIG. 2. Photograph of the chromatogram, run in B.F.W., of the oxidation of quinic acid after 25-30 hours (upper portion) and after 40-42 hours (see text). s a. o a 8 42- a;

132 Hulme and Arthington The Oxidation of Quinic Acid Preparative scale oxidation of quinic acid. Early chromatograms were obtained using approximately 20 mg. quinic-acid and 1 ml. of H 2 O 2 refluxed at no C. for periods up to 3 hours. It was found unsatisfactory to increase this scale directly since the H 2 O 2 was apparently fairly readily destroyed and the resultant water so diluted the reaction mixture that the rate of reaction was seriously reduced. It was therefore necessary to add H 2 O 2 by stages. Nine grammes of quinic acid were refluxed with 25 ml. of H 2 O 2 at 120 C. with the further addition of H 2 O 2 from time to time to a total of 55 ml. Fig. 2 (upper part) shows paper chromatograms run in B.F.W. after 25-30 hours' reaction and after 40-42 hours (upper part). It will be seen that at 42 hours (the reaction was stopped after 44 hours) the reaction liquid contained chiefly two acids, citric acid and the unknown ('X') acid, with the latter predominating. It is interesting to note that during the oxidation the reaction liquid became and remained bright red so long as quinic acid was present; as soon as chromatograms showed this acid to have disappeared the reaction liquid became practically colourless. No explanation of this phenomenon has been found. At the end of the oxidation period the liquid was boiled vigorously without the reflux condenser, to remove as much remaining H 2 O 2 as possible, and then diluted to approximately 2 litres. The titratable acid present was 2*5 g. calculated as citric acid. The liquid was passed down a column containing 71 g. (air-dry weight) of deacidite E (70-100 mesh) at a rate of approximately 50 ml. per hour. After washing the column the acids were displaced by o-i N. HC1 (60 ml. per hour), the displacement liquid being collected in several fractions. Citric and 'X' acid were present only in the last few fractions containing also traces of HC1. For further fractionation these fractions were combined and evaporated to dryness in vacuo at room temperature to remove HC1. The yellowish semi-solid residue weighed 225 mg. It was dissolved in 50 ml. of distilled water and the two acids present fractionated on a small column of Dowex 2 (2 g. dry weight). Much of the displacement liquid (by o-i N. HC1) from this column contained both citric and 'X' acids, but paper chromatograms showed that the last 8 ml. before the HC1 broke through contained 'X' acid and this liquid was evaporated to dryness in vacuo to remove any traces of HC1. It was then taken up in a little warm water and shaken with charcoal. After nitration and evaporation of the filtrate again to dryness in vacuo the almost white solid was recrystallized. It had a melting-point (uncorrected) of 132 0 C. and formed a />-nitrobenzylbromide derivative having a meltingpoint of 84-5 C. On running chromatograms of the acid mixed with pure malonic acid (in both B.F.W. and E.Am.W.) a single spot only was obtained in each case. It was concluded, therefore, that 'X' acid was, in fact, malonic acid. Oxidation of citric acid. Twenty milligrammes of citric acid were refluxed with 1 ml. of H 2 O 2 under the same conditions as used in similar scale experiments with quinic acid. Chromatograms prepared in B.F.W. from spots of the reaction mixture were similar to those shown in Fig. 1, acids D to G. The

Hulme and Arthington The Oxidation of Quinic Acid 133 general picture suggested that acids D to G (Fig. 1) represented stages in the oxidation, of citric acid and that none of these acids was involved in the oxidation of quinic to citric acid (stage 1). A chromatogram run in E.Am.W. of the oxidation mixture of citric acid after 3^ hours' refluxing is shown in Fig. 3, which also includes marker-spots 1 o? o 2 O 3 4 <= O cr c HMAL FIG. 3. Chromatogram, run in 'E.Am.W.', of the oxidation liquid of citric acid (X in the figure). Marker spots of citric (HC) and malonic (H Mai) acids are also shown (see text). of citric and malonic acid. Presumably acids 2 and 3 correspond as a pair to acids E and F on the B.F.W. chromatogram. DISCUSSION The R F and R M values of the seven acids represented by the spots in Fig. 1 are given in Table I. We have not found it possible to obtain exactly consistent values for the R F s of organic acids on different chromatograms unless such elaborate precautions are taken as to render the method tedious in routine work; indeed, provided marker-spots of known acids are run on chromatograms, this is no serious disadvantage. Nevertheless the R M values in Table I are of interest since AR M values in a series remain reasonably constant even when the absolute R F values are varying to an appreciable amount. The significance of the approximately constant value of &R M in the present series cannot be assessed so long as the identity of some of the members is unknown. Acid A (quinic acid) B C D (citric acid) E F G (malonic acid) TABLE I RF value o-i8 0-24 031 0-40 0-47 056 063 Rat value +o-66 + 0-50 +o-34 +0-19 +0-052 o-io 0-24 &R11 Acids B and C remain unknown although one of them may be citricdialdehyde. The identity of acids E and F is also conjectural, but it is probable (from e.g. Deniges Test Deniges, 1902) that one is acetonedicarboxylic acid. Both have an ephemeral existence, F disappearing more rapidly than E. 0-16 0-16 0-15 0-14 0-15 0-14 X HC

134 Hulme and Arthington The Oxidation of Quinic Acid The existence of acetonedicarboxylic acid as an intermediate in the oxidation of citric acid (and, consequently, of quinic acid) is corroborated by the universal appearance of acetone (detected as the 2:4-dinitrophenylhydrazone) in the oxidation products of both acids. Malonic acid on oxidation breaks down to acetic acid and considerable amounts of acetic acid were found in the evaporates of the quinic and citric oxidation liquids. This breakdown to acetic acid is undoubtedly a contributing cause of the very low final yield of malonic acid and may account for malonic acid not being previously reported as an in vitro oxidation product of citric acid. It has therefore been firmly established that peroxide oxidation of quinic acid yields citric and malonic acids. In addition there is strong presumptive evidence that acetonedicarboxylic acid is formed during the conversion of citric to malonic acid. In an earlier paper (Hulme, 1951) the direct aromatization of quinic acid in plants, possibly via shikimic acid, to such benzenoid compounds as protocatechuic acid has been discussed. Although such reactions are known to occur in micro-organisms, their widespread occurrence in vegetative organs of the higher plants is not proved. In the literature on plant biochemistry and physiology from the time of Goiter (1909) the widespread occurrence of the depside chlorogenic acid has been reported. Gorter showed 'chlorogenic acid' to be composed of one molecule each of caffeic acid and quinic acid. Recent work has shown the presence of chlorogenic acid-like compounds in various plant tissues (e.g. in the sweet potato by Rudkin and Nelson, 1947), but the results of the application of the modern techniques of partition chromatography suggest that 'chlorogenic acid' as used by some of the older workers is a general term for several closely allied depsides. Free quinic acid may, however, act as a source of citric (or even malonic) acid, and although Hall (1937) suggests that plants noted for their quinic acid content are not producers of citric acid, we have some evidence that citric acid appears in mature apples. Mr. L. S. C. Wooltorton carried out most of the practical work involved in this paper. The work described in this paper was carried out as part of the programme of the Food Investigation Organization of the Department of Scientific and Industrial Research. {Crown Copyright Reserved) LITERATURE CITED DENIGES, M. G. (1902). Les sels mercuriques comme r actifs en chimie organique. Bull. Soc. Chim. de Paris, 3 me se>. 27, 13. FISCHER, H. O. L., and DANGSCHAT, G. (1934). Abbau der Chinasaure zur Citronsaure. Helv. Chim. Ada, 17, 1196. GORTER, K. (1909). Ueber die Verbreitung der Chlorogensaure in der Natur. Arch. d. Pharmazie, 247, 184.

Hulme and Arthington The Oxidation of Quinic Acid 135 HALL, J. A. (1937). A system of structural relationship in phytochemistry. Chem. Rev. 20, 305- HII&T, E. L., and JONES, J. K. N. (1949). Quantitative analysis of mixtures of sugars by the method of partition chromatography. Part III. Determination of the sugars by oxidation with sodium peroxide. J. Chem. Soc. 1659. HULME, A. C. (1951). The isolation of /-quinic acid from the apple fruit. J. Exp. Bot. 2, 298, 1951. LONG, A. G., QUALE, J. R., and STEDMAN, R. J. (1951). The separation of acids by paper partition chromatography. J. Chem. Soc. 2197. LUGG, J. W. H., and OVERELL, B. J. (1948). One- and two-dimensional partition chromatographic separations of organic acids on an inert sheet support. Aust. J. Set. Res. A. 1, 98. RUDKIN, G. O., and NELSON, J. M. (1947). Chlorogenic acid and respiration of sweet potatoes. J. Amer. Chem. Soc. 69, 1470.