ROBERT R . GRINSTEAD
3472 [CONTRIBUTION FROX
THE
RESEARCH DEPARTMENT, WESTERN DIVISIOX,THE
Vol. 82 IIOW
CHEMICAL CO., P I T T S B C R G , CALIF. 1
Oxidation of Salicylate by the Model Peroxidase Catalyst Iron-Ethylenediaminetetraacetato-iron(II1) Acid BY ROBERTR. GRINSTEAD RECEIVED DECEMBER 14, 1959 In the presence of ethylenedlaminetetraacetato-iron( 111) and ascorbic acid (model peroxidase system) salicylate is osidized in aqueous solution by 0 2 and Hz02. The principal products are 2,3- and 2,5-dihydroxybenzoic acids. Yields, based on salicylate consumed, ranged from 50-70%, and t h e ratio of 2,3- t o 2,5-products varied from 1.1 t o 3.2. Hzoz appears t o b- a n intermediate when 0 2 is the oxidant. Benzoic acid was oxidized t o a mixture of o-, m- and p-hydroxybenzoic acids. T h e d a t a are discussed in terms of a free radical mechanism, wherein initiation occurs by reaction of HZOZor 0 2 with the ferrous chelate, producing HO. or H01. radicals. Reaction of these radicals with salicylate produces a n aryl radical which continues the chain and leads ultimately t o the dihydroxy acid.
The “model” peroxidase system1**is of interest because of the close similarity between its behavior and that of the enzyme peroxidase. I n the presence of oxygen and a reducing agent possessing an ene-diol structure,
-COH
11
both systems hydroxyl-
-COII
ate aromatic compounds a t specific sites on the ring,2-6 and in both cases the entering hydroxyl is derived from the oxygen m o l e c ~ l e . ~ JThis type of activity is not peculiar to peroxidase, but is exhibited by a number of other enzyme systems, both in oitro and in vivo. An extensive review of this subject has been given by Mason.7 Because of its greater simplicity, a knowledge of mechanism of action of the model system is important, not only because of the light which might be shed on the enzyme mechanism, but also because of the potential utility of the model as a preparative method for phenolic compounds. Because of the apparent participation of hydrogen peroxide as an intermediate in this reaction, the mechanism of the H20zascorbic acid reaction was studied first. I n the preceding paper,6the kinetics of this reaction were examined, and a free radical mechanism was proposed which accounts for the observed behavior. In this paper, the behavior of salicylic acid in this system (and in similar systems utilizing oxygen as the oxidant) is presented. This aspect of the work was confined mainly to isolation and identification of the products formed under various conditions.
was checked at dilutions of 12,500 and 125,000 in a solution containing 0.1 M phosphate, and 1.1 X 10-3 M H2Oz at a pH of 6.5. The initial rates of H2O1decomposition were, and 5 x 10-4 mole per liter per respectively, 5 x minute, Procedure.-The course of a n experiment was followed in the cases where H202 was the oxidant by dilution of a n N HzS04 t o a known volume in 0.04 aliquot with 1 Ti(1V) and determining the optical density of the solution a t 410 mp. When oxygen was used as a n oxidant the reaction was carried out in a constant volume system containing pure oxygen, equipped with a manometer and a magnetic stirrer, I n experiments where ascorbic acid wds present, the reaction with HZO2was over within a few minutes, a t which time the H202had been completely consumed. The reaction with O2 required somewhat longer, b u t was complete within 3 to 4 hours and continued until the ascorbic acid had been consumed Analysis of Products.-Unreacted salicylic acid was removed from the reaction by acidification and extraction with chloroform. Further extraction with ether gave a mixture of the dihydroxybenzoic acids. These were not separated further, but were identified by three different methods. The infrared spectra of the mixtures were recorded and compared with known samples of the various suspected products. Besides serving to identify the products, it could be shown also t h a t salicylic acid was effectively removed by the preliminary chloroform extraction. As a check on the infrared scans, the ether residues were paper chromatographed on Whatmarl No,: 1 filter paper, using 4:l ( b y volume) i-propyl alcohol-i N “,OH as the eluant,g and developing with 1% ferric ferricyanide solution. Ri values determined in this system arc
Experimental Materials.-Salicylic acid (USP) was made u p as a solution with the buffer prior to use. The ascorbic acid used W A S T3P grade. Other chemicals were reagent grade. Rextions were carried out by mixing the appropriate solut i m s , adding the Hz02 last. Oxygen was generally excluded from the system during a run except where i t was sp-cifically involved as the oxidant. Catalase was obtained as a solution of unspecified concentration from the H;AI Chemical Co., Santa Monica, Calif. The activity ~-
I I ’ S . Udenfried, C. T. Clark, J . Axelrod and B. B. Brodie, J . Bid. r h ? 1 7 . , 208, 731 (1954). ( 2 ) B . B. Brodie, J. Axelrod, P. A. Shore and S. Udenfriend, ibid., 208. 7 4 1 (1954). ’3) C . E. Dalgliesh, Avch. Biocher?~. B i o p h y s , , 58, 214 (1955). ‘41 H. S. Mason, I. Onoprienko and D. Buhler, Biochim. et Biophys. .4cta, 2 4 , 223 (19b7). i.3) H S Mason, I. Onoprienko, K.Yasunobu and D. Buhler, THIS
79, 5578 (1957) ’ 11. S hlason and I . Onoprienko, F r d e r a t i u n Ptor., 16. 810
;OL-RSAL,
’ H. S l f a s o n , A d u . Eirzymol., 19, 7G (1057). (R) R R.Grinstead, T H I S Joun.v.4~,82, 3464 (19GO).
Acid
Ri value
2,6-Dihydroxybenzoic 2,5-Dihydroxybenzoic 2,4-Dihydroxybenzoic 2,3-Dihydroxybenzoic o-Hydroxybenzoic m-Hydroxybenzoic p-Hydroxybenzoic
0.71 45 ,22 .33 .68 .30 .16
Actual analyses of the ether fraction were made by two methods. The 2,3-dihydroxybenzoic acid was determined by the colorimetric method given by Snell and Snell,*O involving the color developed by alkaline ferrous tartrate and catechols. The total o-hydroxybenzoic content was determined by a second colorimetric procedure” utilizirig the color developed with ferric chloride. Because or~l?. two constituents were present, these two values allowed a determination of the composition of the product t o be made. In one experiment benzoic acid was used as the substrate. The separation was carried out in a similar manner. The chloroform residue, which was mostly unreacted benzoic acid, also contained some salicylic acid, which was a (9) R . J. Black, E. L. Durrum and G . Zweig, “Paper Chromatography and Paper Electrophoresis,” 2nd ed., Academic Press, Inc.. SPIV York, S‘ Y . , 1958, p. 307. (10, F D Snell and C T Snell, “Colorimetrlc h l e t h o d s of Anal?. his.” Vol. 111, 3rd ed., Van Sostrand Co , Inc , F e w Vork, S-. I’.,19.3, p . 127.
(11) Reference 10, p. 4 2 4 .
PEROXIDASE OXIDATIONOF SALICYLATE
July 5, 1960
3473
TABLE I MODELPEROXIDASE SYSTEM.OXIDATIONOF SALICYLIC ACID BY HzO?AND O? M , [ascorbic a r i d ] = 0.10 hf [Phosphate] = 0.10 M , [salicylate] = 0.10 M , [Fe] = 1.0 X lO-'M, [ E D T A ] = 1.0 X Expt.
PH
Conversion of Yield of dihydroxys, yo [ H ~ O ~ ] , salicylate, --based on----M %" Salicylates HnOnC Ond
4.3 0.10 76-62 .10 6.5 i6-92 Oe 6.5 71 0' 6 5 98' 7, salicylate reacted. Moles product Moles product found/2 X moles 0 2 used.
Ratio 2,3:2,5
Product composition, % -(ether residue)-2, 3 2, 5 Total
3.2 $0 22 92 30 55 16 17 2.2 65 29 94 27 60 28 61 14 1.8 60 34 94 1 1 51 45 96 30 67 15 found/moles salicylate reacted, c Moles product found/moles H202 reacted. e OZused as oxidant. f Catalase solution added a t a dilution of 1:SO
product of the reaction. This was determined analytically as before. The ether phase was paper chromatographed in the same solvent as above, and the presence of both mand p-hydroxybenzoic acids was established. Semiquantitative estimates of these compounds were made by colorimetric methods. The p-OH isomer was estimated with Millon reagent12 and the total phenols with p-nitroaniline.I3 Some dihydroxy acids were detected also by the procedure described above.
presence of the enzyme peroxidase and the enediol dihydroxyfumaric acid HOO>C=C
