heating, some inaterial had appeared 0 1 1 the cooler parts warniiiig O m drop of concentrated sulfuric acid was of the flask. Further heating had no effect. The sub- added and the solution warmed for a few minutes longer, limate was then scraped from the walls of the flask and then allowed to cool. After two hours a small amount of recrystallized from ethanol. Only a few milligrams of precipitate had appeared. This was filtered off and reproduct melting at 236-237" was obtained for each gram crystallized from dilute methanol; there was not enough of 111 used. Admixture with an authentic sample of s-di- material for two recrystallizations. There was obtained a 1)henylurea produced no depression of the melting point. white powder, m. p. 148-1,507°: admixture with XIT g a w Conversion of V into XU--Two grams of V was dis- a melting point of 152-164 . wlved in 125 cc. of dioxane and 10 cc. of water and 10 c ~ . of- concentrated hydrochloric was then added. There summarg was immediate formation of a yellow color upon addition 1. I t has been shown that the reaction uf of the acid. After standing for twenty-four hours, thr acetaldehyde with ethyl 0-anilinocrotonate yields holution was poured into 300 cc. of water. After standing for forty-eight hours, the suspension a dihydropyridine derivative. resulting from this treatment had deposited yellow leaves. 2. It has been shown that the reaction of These were removed by filtration and washed with water. benzaldehyde with ethyl p-anilinocrotonate yields There was obtained in this maimer 0.70 g. of dry yellow product, m. p. 1fX-176". Two recrystallizations from a dihydropyridine derivative and a tetrahydromethanol gave pink nwdles melting a t 175-177 ~ ' , pyrimidine derivative and that the reaction of T h e pink color accompaiiying this product is as difficult benzaldehyde with methyl 6-anilinocrotonate to remove as that accompanying XI ; its removal was accomplished by use of the same techiiique as that previously yields a tetrahydropyrimidine derivative. 3 . Several degradation products of these tetradescribed for XI. This treatment gave very nearly white needles, m. p. 177-178" hydropyrimidines have been isolated. The ac-4nal. Calcd. for C&diOa: C, 77.90; H , 6.01; S , tion of dilute hydrochloric acid upon these tetra3.63. Found: C, 78.00; H, 6.74; S , 3.71. hydropyrimidines yields compounds which are Synthesis of =I.-Four-tenths gram of methyl a- probably oxazines. acetyl-8-anilinohydrocinnatnate and 0.50 g. of benzaldeIiydc were mixed and disqolved in 1 cc. of methanol with MIWEAPOLIS, MINNESOTA RECEIVED MAY21, 1945
[ C u S y ' R I B U T I O x F R O M 'THE I)I\vISIOS O F PfIYSIOLOGICAI, CHEMISTRY, c : S I V E K S I T Y OF
MINNESOTA, MINNEAPOLIS]
Spectrophotometric Studies of the Oxidation of Fats. 11. The Oxidation of Dienoic Fatty Acids BY RALPHT. HOLMAN,' WALTER0. LUNDBERG~ AND GEORGE 0. BURR The changes in fats which take place during autoxidation have been the subject of much study and many workers have proposed mechanisms for the oxidation of the component unsaturated fatty acids. Von Mikusch and Priest3 have summarized briefly the theories and proposed mechanisms by which oxygen may attack the double bond. The mechanism whereby a fourmembered ring peroxide is formed is favored with the more unsaturated fatty acids. The demonstration of oxido structures among the products of autoxidation of monoethylenic systems by Ellis4 and Deatherage and MattilP lends support to the mechanism leading to a threemembered ring peroxide containing tetravalent oxygen. Fahrions first proposed a rearrangement of peroxides to form a keto1 and MorrelP and co(1) This paper represents part of a thesis presented by Ralph T. Holman t o the Graduate Faculty of the University of Minnesota in partial fulfillment of the requirements for the P h . D . degree, June, 1944. The work was aided by grants from the Hormel Research Foundation, the National Live Stock and Ment Board, and the Sational Dairy Council. (2) Present address: Hormel Institute, Austin. Minaesota. 13) J. D . Von hIikusrh :and G . W Prie.st. Oil nird Ton,+ 18, XI I
1941). (4) G . W. Ellis, Biochrm. J , SO, 757 I lY:36). ( 5 ) F. E. Ueatherage ani1 FI , \ Xlattrii. I n d . b.tig.
( I>?,,? 31, 1426 (1931). (6) W.Z.Fahrion, Angew. Chew,., 16, 665, 697 (1903). (7) B. S . Morrell and E . 0. Phillips, Feltr u . S e i f e n , 46,541 (1939).
workers have been able to identify ketols as products of oxidation of p-eleostearin. EllisBsuggested that the addition of oxygen to the double bond yields an ene-diol. Scheiberg postulated the addition of oxygen to the unconjugated diene system a t the active methylene group to form -CH===CH-CO-CH=CH--. Farrnerlo~ilproposed a mechanism whereby oxygen attacks the carbon between the double bonds of the linoleic acid type system to yield a hydroperoxide. It will be noted that in the last three mechanisms the system retains its unsaturation after the addition of oxygen. In disagreement with these mechanisms is the work of Paschke and Wheeler12who found the decrease in iodine value roughly parallel to the increase in peroxide value during the oxidation of fatty acid esters. Some work has been done relative to the spectroscopy of fat oxidations. Edisbury and c o - ~ o r k e r found. s ~ ~ an increase in the absorption of ling oil upon its exposure to air. In the oxidized sample there was an indication of an absorpi8) G. W.Ellis, J. SOC.Chcm. Ind.. 46, 193T (1926). (9) J. Scheiber, Z. nngcw. Them., 46, 643 (1938). (IO) E. H.Farmer, G. H. Bloomfield, A . Sundralingam and D. .k. Sutton,,Trans. Faraday SOC.,88, 348 (1942). ' 11) 13. 11. Farmer and D. 4 . Sutton, J , Chcm. SOC.,119 (1943). I 12) K.P. Paschke and D. H. Wheeler, O i l atid Soap, 21, 62 (1941). (13) J . K. Edisbury, K -4, hIorton and J . .4. Lovern. Biochem. J., as, 899 (1936).
THEOXIDATION OF DIENOIC FATTYACIDS
,lug., 1045
1387
2.0
1.o 1 .o
-1.0 -1.0
-2.0
-2.0 2500
3000
3500
4000
A. Fig. 1.-Ultraviolet absorption spectra of ethyl linolate: (I), ethyllinolate, P.V. = 5.6; (2), (1) in KOH; (3), ( 1 ) in air R3', 70 hr., P.V. = 211.0; (4),(3) in KOH.
tion band near 2700 A. Farmer's group" reported a general rise in the absorption spectrum of methyl docosahexaenoate with the disappearance of the band a t 2700 A. and an increased absorption a t 2300 A. when the substance was exposed to air. In a subsequent study Farmer and co-workers16 repeated this work with the same result, and also reported that the absorption bands a t 2300 and 2700 A. in ethyl linolenate increased upon oxidation. A comparison of squalene and oxidized squalene showed a significant rise in the absorption upon oxidation, but no pronounced band appeared. From their studies these workers concluded that oxidation of unsaturated fatty acids is accompanied by conjugation of the isolated double bonds. Mitchell and Kraybilllb found that at the beginning of oxidation the absorption due to conjugation caused'by bleaching, is reduced; however, as had been previously shownldbfurther oxidation of linseed oil is accompanied by increased absorption which does not have the fine structure characteristic of triene conjugation between carbon atoms. Brauer and Steadman17 studied the course of the oxidation of /3-eleostearic acid and found that as the oxidation proceeded the band a t 2680 A. dis(14) E. H. Farmer and D. A. Sutton, J . C k m . SOL,122 (1943). (15) E. H. Farmer, H. P. Koch and D. A. Sutton, ibid., 541 (1943). (16) (a) J. H. Mitchell, Jr. and H. R. Kraybill, THISJOURNAL, 91, 988 (1942); (b) I n d . Eng. Ckcm.. Anal. E d . , 18, 785 (1941). (17) R. W. Brauer and L. T. Steadman, THISJOURNAL, 66, 563 (1944).
~
~~
2500
~
3000
3500
4000
~~
4500
A. Fig. ?,.-Ultraviolet spectra of ethyl linolate: (1). ethylliiolate, P.V. = 5.6; (2), (1) in KOH; (3), (1) in air IIO", 192 hr., P.V. = 38.3; (4),(3) in KOH; (5), (3) in vacuum l l O o , 312 hr., P.V. = 0.5; (6), (5) in KOH.
appeared. Since conjugated systems with accompanying light absorption may be produced in fats by saponification, by distillation,20by debromination,21 and by treatment with adsorbents,16as well as by oxidation, the appearance of weak bands in the spectra of fats need not be directly related to oxidative changes. It was shown in the first part of this study22that upon oxidation of oleic acid, ethyl oleate, and elaidic acid the absorption increased with a marked inflection a t 2750 A. and a maximum a t 2300 A. The following pages describe the changes in absorption spectra of the doubly unsaturated fatty acids.
Ekperimental The ethyl linolate used in this study was prepared by the debromination procedure of Rollet.*8 The sample boiled at 180' (2 mm.) and showed a peroxide value of 5.6. The linoleic acid" used was prepared by repeated low temperature crystallization. Crotylideneacetone was prepared by the method of Meerwein*&( ~ Z I ~ 1.5136), D and mesityl (18) T. Moore, Biochcm. J . , 88, 1635 (1939). (19) J. P. Kass and G. 0. Burr, THISJOURNAL, 61,3292 (1939). (20) F.A. Nerris, I. I. Rusoff, E. S . Miller and G. 0. Burr, J . Biol. Chcm. : 147, 273 (1943). (21) W. R. Brode, J. W. Patterson, J . H. Brown and J . Frankel, Ind. Eng. C h m . , Anal. E d . . 16, 77 (1914). (22) R. T. Holman, W. 0. Lundberg, W. M. Lauer and G 0. Burr, THISJOURNAL, 67, 1285 (1945.) (23) A. Rollet, Z . pkrsiol. Chcm., 64, 410 (1909). (24) Kindly supplied by Dr. J. B. Brown. (25) € Meerwein, I. Ann., 866, 85 (1909).
Vol. F7
RALPHT. HOLMAN, WALTER 0. LUNDBERG AND GEORGE 0. BURR
1388
2.0
I .D
6p& c 3
Y M
O
-1.0
Fig. 3.-Ultraviolet absoxption spectra of linoleic acid: ( I ) , linoleic acid by low tem2erature crystallization; ( 2 ) , (1) in KOH; (3), (1) in air a t 63", 96 hr., P.V. = 280.0; (4), (3)in KOH. oxide and phorone were prepared by the method of Bayer.*' p-lonone was purchased from du Pont and acrolein and a-methylacrolein were purified from the Shell product. The lO,l2-linoleic acid was prepared by Dr. J. P. Kass. T h e methods used in this study are the same as those in the study of the monoethenoic acids.2e
Results and Conclusions The sample of ethyl linolate used in this study unfortunately was not pure as shown by the peroxide value of 5.6. Its absorption spectrum showed a band in the region where conjugated trienes absorb strongly (Fig. 1). In alkali this band decreased slightly and fine structure appeared, but the absorption inqeased considerably at longer wave lengths with the appearance of a maximum a t 3700 A. Oxidation of the ester for seventy hours a t 63' altered the spectral quality only slightly in either neutral or alkaline solution, except for a rise in absorption a t 2350 A. Treatment of the sample a t 110' for 192 hours, conditions under which peroxides decompose readily and polymerization takes place, increased the absorption a t 2700 A. considerably (Fig. 2 ) . Further heating in vacuuni a t 110' did not greatly change the absorption spectrum. None of the changes in absorption produced by oxidation are closely related to the peroxide value. Linoleic acid, highly purified by twelve re(26) A. Baeyer, Anr., 145, 297 (1866)
2500
3000
350(1
4000
4500
A. Fig. -l.-Ultraviolet absorption spectra of l0,lZ-linoleic acid: (I), l0,lZ-linoleic acid; (2), (1) in KOH; (3), (1) in air 63', 45 hr., P.V. = 381.0; (4), (3) in KOH.
crystallizations a t low temperatures, showed almost identical spectra in alcohol and in alkali (Fig. 3). This indicates that cool alkaline solution has no effect upon the unconjugated doubly unsaturated system. Upon oxidation the acid showed a marked increase in absorption and the absorption in alkali a t 2750 A. was almost double that in alcohol, indicating that the band a t 2750 A. was due in part to enolizable substances (Table I). Conjugated linoleic acid, whose spectrum is unchanged by alkali, becomes sensitive to alkali after oxidation (Fig. 4). The absorption a t 2350 A. due to the conjugated diene system decreased markedly with oxidation while the absorption above 2500 A. increased. A band appeared a t 2750 A. which was accentuated in alkali. A pronounced band a t 3800 A. also appeared in the alkaline oxidized sample. I n summary, Figs. 1, 2, 3 and 4 show that like fresh oleic acid, the absorption spectrum of unoxidized linoleic acid (both natural and conjugated) is unaffected by alkali. However, the products of autoxidation behave wholly unlike those of oleic acid in that the absorption a t 2700 A. is only slightly affected by alkali while a pronounced band is produced a t 3700 A. The type compounds used for the study of oleic acid and its oxidation products22 included di-
1389
THEOXIDATIONOF DIENOIC FATTYACIDS
Aug., 1945
TABLEI
ABSORPTION MAXIMAFOR SAMPLES OF FRESH AND OXIDIZED LINOLATES KOH
Alcohol ___c-
Treatment
Substance
Ethyl linolate
1) Fresh
2) (1) 63",70 hr. 3) (1)l l O o , 192 hr. 4) (3)vacuum llOo, 13 days Linoleic acid
5) Fresh (5)63",96 hr.
G)
10,12-Linoleic acid
7) Fresh
P.V.
Amax.
2700 A.
2.39
211.0
2700
2.03
38.3
2300 2700 2300 2700 2750 2725 2775 2300
98.0 17.4 65.7 16.7 0.38 12.3 12.2 916.0 725.0 107.0 6.30
5.6
0.5
... n9.0
...
2350"
8) (7)63",69 hr.
Emu.
381.0
2300 2750"
Amu.
2575 A. 2675 2800 3650 2700 3600 3700" 2800 3700" 2750 2750
2350 2750 3700"
Emex.
0.97 1.08 0.95 0.27 1.48 0.19 3.42 26.0 5.34 0.34 21.8 591.0 28.3 4.10
" Denotes inflection. hydroxy-, ketol-, diketo-, oxido-stearic acids, and their autoxidation products. In addition, ace-
Since it has been postulated that linoleic acid may add oxygen without destruction of the double
4 4.0
3
3.0 2 4
s"
u' M
3 2.0 1
1.0 0
2500 3000 3500 4000 4500 5000
A. Fig. B.-$pectra of reference compounds: (I), acrolein in alcohol; (2), (1) in KOH; (3), a-methylacrolein in alcohol; (4),(3)in KOH;(5),mesityl oxide in alcohol; (6), (5)in KOH.
0
2500 3000 3500
4000 4500 5OOO
A. Fig. G.--Spectra of reference compouuds: (l), 8ionone; (2), (1)in KOH; (3),crotylideneacetone; (4), (3)in KOH;(5),phorone; (6),(5)in KOH.
tone, acetonylacetone, acetylacetone and diacetyl were studied. None of these have spectral quali- bond (see above) other type compounds were ties in neutral and alkaline media similar to those studied in neutral and alkaline alcohol. These of autoxidized linoleic acid. were :
Acrolem n-Methylacroleit: 5Iesityl oxidt Phorone ~rotylidcr~eacrtori~
CH.=CHCHC, CH;z=CH(CHy)CHO 'CHj)?C=CHCOCH (CHIIJC -CHCOCH=X(CH j I CH CH - C H C H CFTCOCH H C
c
H (
,~-IonoliL
I'TI i CH
(
HCOC"
.In exaniiiiatioii oi Figs. 5 aiid (ishows that the absorption spectra of these substarices are iiot much like those of oxidized linoleic acid. Special attention should be called to phorone and crotyildeneacetone. 'The former has the structure postulated by ScheiberYfor ail oxidation product of linoleic acid. Only iii alkaline solution does oxidized linoleic acid exhibit broad bands at 2700 and 3700 A. much like those of phorone but of different relative intensity. The bands of phorone and crotylideneacetorie are so iiitense that a fraction of one per ceiit. could account for all of the absorption a t 2700 A. shown by the most highly oxidized linoleic acid used in this study.
[CONTRIBUTIOX FROM
Summary 'The absorption spectra of ethyl liiiolatc, highly purified linoleic acid, and 10,12-liiioleic x i t i were studied before and after oxidation in iieutral :iiid alkaline alcoholic solutions. 2 . .lutoxidatioii of each of these compounds is >tccotiipaitied by an increase i i i the absorptioii at 27.50 A. which is not directly related t n peroxide vnluc. 3. ( kitlatioil of conjugated 1i;ioleic acid greatly reduces thc absorption at 2300 A . 4. Cool alkali has no effect upoil the absorption of the conjugated or unconjugated fresh fatty acids but produces market1 ch:mges in the oxidized products. .-I. ;ibsorption spectra of acrolein, a-methyl acroleiii, mesityl oxide, phorone, crotylideneacetone, and (3-ionone in alcohol and alkali 'are shown as examples of unsaturated conjugated carbonyl compounds. (j. The autoxidation products of linoleic acid are spectroscopically unlike those of oleic acid. I.
~ ~ I S Y E A P I : I I . I1-1, S
THE' D I V I S I O N OF PIIYSIOLOGICAL
CHEMISTRY,
bhSX.
RECEIVEDMARCH 1-1, 194-5
c % I V E R S I T Y OF MINNI.:SOTA]
Spectrophotometric Studies of the Oxidation of Fats. 111. Ultraviolet Absorption Spectra of Oxidized Octadecatrienoic Acids BY RALPHT. HOLXAN,' WALTER0. LUNDRERG~ AND GEORGE 0 . BURR
In previous investigations3s4 the changes of ultraviolet absorption spectra were followed during the courses of oxidation of fatty acids containing one or two double bonds. The present report presents the changes in spectrum induced by the oxidation of the fatty acids containing three double bonds, isolated or conjugated. Experimental The ultraviolet absorption spectra were determined using the Beckman quartz spertrophotometer. 98'; ethyl alcohol distilled over potassium hydroxide was used as solvent. After the spectra in alcoholic solutions w e n determined, 5.0 cc. of the alcoholic solutions were diluted hydroside, aiid the with 5.0 cc. of ZOi,;, aqueous potassiu~r~ spectra in alkali determined inmediately. Linolenic acid and ethyl linolenatc wcrc exposed to air in ail oven at G3", the temperature used iri an oven A h bility test! A second samplc of litiolenic acid which h a d (1) This paper is taken in part from a thesis presented by Ralph 'T. Holman to the Graduate Faculty oi t h e University oi Minnesota in partial fulfillment of the requiremeuts for t h e Ph.1). degree. June. 1944. The work was aided by grants from thewormel Research Foundation, t h e National Live Stock and Meat Board, aud t h e S a tional Dairy Council. (2) Present address: Hormel Institute, Austin, Minnesota. (3) R. T. Holman, W. 0. Lundberg, W. M ,1,auer a n d G . 0.Burr, TriIs J O U R N A L , 67, 1285 ( 1 9 4 5 ) . (4) R. T. Holman, W'. 0 1,undt)erX nnri C: 0. Hurr, %bid . 67, 13% Il045) $ 5 ) IV. 0. L u n d b r r g , I i ( 1 l l a i ~ ~ , r . ~ i
