Cyclopropenoid Fatty Acids Arthur Greenberg and Joseph Harris Department of Chemical Engineering and Chemistry, New Jersey Institute of Technology, Newark, NJ 07102
A cyclopropene ring, one of the most strained structures in organic chemistry, is a surprising unit to find in a naturallyoccurring molecule. Nevertheless, during the 1950's two fatty acid, acids, sterculic acid (2-octyl-1-cyclopropene-1-octanoic 1) ( I ) and malvalic acid (2-octyl-1-cyclopropene-1-heptanoic acid, 2) (2) were discovered to he significant components of oils from seeds (and other tissues) of plants in the order Maluales (3-6) which includes cotton. How do cyclopropene rings come to be in natural molecules, and what "havoc," if any, do these reactive units create in living organisms? Introductory organic chemistry texts which mention these fatty acids do little more than include them as natural oddities. While this is understandable, it is unfortunate since, as we shall demonstrate, a more detailed investigation will entail explorations into the realms of novel aromatic structures. caibene chemistry, enzymology, toxicology, and food science. It represents a golden opportunity to demonstrate to students of the life sciences that even a hit of seemingly purely chemical exotica snch as cyclopropene can he very relevant to their interests. CH2
\
cHmuj6/=cm,,,cofi I
CHs
cH,(cH,)i6/=c(cH,~co?H \ 2
Two plants in the order Maluales play particularly important dietary roles: the cotton plant (Gossypium hirsutum) furnishes an oil present in many American food preparations while oil from kapok seeds (Eridendran anfractuosum) is important in oriental diets. Cottonseed meal, which is accompanied hy the oil, is a major protein supplement for farm animals as well as commercial fish snch as rainhow trout. Although malvalic acid is usually three to five times more abundant than sterculic acid and the total cyclopropenoid fatty acid (CPFA) content of plant oils is typically 1-2.570, the fatty acids of the seed oil of Sterculia foetida are 50-70% cyclopropenoid and the sterculic acid-to-malvalic acid ratio is 10:1(5). (7. Cvclourouene . . . manifests a number of unusual ~ronerties . . 81 including a high dipole rntment 10.455 T)I tora h?dncarh;ni 10.1111. high rri~utivitvt u w ~ r dadditlon rcnrtionsdri\cn 1n.a 26'kcallmh reduction in strain energy npon conversion a cyclopropane derivative as well as a tendency to complex with metals ( l l ) , and ring-opening reactions which sometimes involve vinyl carhenes (12). I t is interesting to investigate whether these properties exert profound effects on biological svstems. Furthermore, it is worth noting. that althoneh cvciopropenes were first synthesized dur& the 1920's (131, these earls syntheses confirmed nearly twenty years later (14). and an electron diffraction structure of c~ciopropenepuhlished in 1952 (151,postulation of a cyclopropene as a mechanistic intermediate could still invite derision in the mid1950's (16).
action is hdicated by H pink or red color. Subsnluently. t h ~ : test underuent a bewilder:np number of modilicatir,ns !:I,. and it will he discussed later in chis article. In 1928, cottonkid oil incorporated into the diets of hens was shown to cause the whites of their eggs to turn pink ("pink white disorder") and their yolks to assume a bronze color and pasty texture (18). In the late 1920's and 1930's a number of other oils from plants of the Maluales family were also shown to give positive Halphen tests (3). A crucial finding was the discovery that only those oils which gave positive Halphen tests were capable of producing "pink white disorder" (19). The origin of the pink color was subsequently shown to he an iron chelate of coralbumin in the egg white caused by increased ability of iron to diffuse from the yolk (20). Sterculic acid was first isolated via 2-methylpentane extraction of the seeds of Sterculia foetida by Nunn who published the correct structure of this substance in 1952 (1)(curiouslv. ,. there are no nrecedents cited in thnt paper for prior characcerizntionsuf cyc1opropt:nes). Although the structure rmvosed hv Nunn was initisllv rhallt~nrd 121 I. " , ,, it soon received overwheiming support (22125). The association of ~ositiveH a l ~ h e ntests with the Dresence of CPFAs was made 1;y two groups in I955 (22,2,51nncl a positive ~ a l p h c n test ~usinaC3, alonr) with sterculirarkl rrvorted in 1956 1261. ~ a l v a l i c & i d w a sisolated in 1956 (2), anb its structurew& reported in 1957 (27). A material tentatively called "bomhacic acid" (22) was later shown to be a mixture of 1 and 2 (28). During this period "pink white disorder" was found to he induced by feeding malvalic acid (27) or sterculic acid (29) to hens. The first reported synthesis of sterculic acid involved Simmons-Smith reaction (CHII?, z i n c - c o ~ ~ eofr )the alkvne precursor steardic nrid (!hctaderynoic itid, 31, in 44 yield (MI.Othrr workers (31I were not able to dunlimle t his synthesis and instead achieved a six-step, 30%yield of methyl sterculate via cyclopropeninm ion intermediates. A related three-step pathway to methyl sterculate (60-65% yield) was published shortly afterward and is summarized in I (32). A similar route was employed for the synthesis of methyl malvalate and other 1,2-dialkylcyclopropenes(33). ~
tb
Historical Background The main impetus which led to the discovery of cyclopropenoid fatty acids came from the food and agriculture industry. The need to detect adulteration of more expensive oils with cottonseed oil gave rise to the Halphen test. As originally uuhlished in 1897 (17). it involved addition to the oil of one ;olume of carhon disulkide contain~ngIC1 freesuliurnnd ouc volunie of nentnnd. 'l'be solution is h r a t ~ dslou~l\~ to I 10°C in an opentube accompanied by loss of CS2, and positive re-
~~~
~
~~~
~
CO.C,H, 4
-
I
Cyclopropenoid fatty acids appear to manifest two types of effects npon eggs. The first is a nonspecific effect upon membrane permeabilitv andlor yolk structure which affects material distribution. Thus, iron migration to the yolk is accompanied hy other changes inclndina transfer of water and protein from~thewhite to-the yolk and nonprotein nitrogen transfer (chiefly in the form of amino acids) from the yolk to the white (5).A more specific effect is the inhibition of fatty acid desatnrase which decreases the yolk content of oleic acid and increases the content of the higher melting stearic acid. This gives rise to the pasty texture of the yolks. Ingestion of Volume 59
Number 7 July 1982
539
CPFA, by cows produces changes in the quality of milk, sticky butter, and hard lard for the same reasons. Selected Chemical Reactions of CPFAs
Sterculic acid polymerizes a t a significant rate at room temperature ( I ) and is much more stable in the form of its methyl ester (33).The polymerization reaction clearly involves ring opening ( I , 23, 34, 35) in contrast to the situation for simple cyclopropenes lacking suhstituents a t the 3-position which undergo ene reactions which maintain the threemembered rings (36). The free carhoxyl group in I apparently induces ring opening. Major products include allylic derivatives of the type 7-10 (R = R' = sterculic acid residues (23,34, 35). CH2
CH2
are thermally accessible from cycloprupenes (39). The formation of small amounts of conjugated dienes from 1,2-din-octylcyclopropene(35)is consistent with some vinyl carhene formation. Furthermore, the formation of conjugated dienes, as the major products of reaction of CPFAs with silver nitrate-silica gel (40) is reminiscent of the silver-catalyzed ring opening of simpler cyclopropenes which is thought to proceed through argentocarbenes (41).
20
22
21
23
Thus, we propose the sequence depicted in 111 as a means of explaining formation of 15 and 16.
I11
The occurrence of these products is readily explicable in terms of the intermediacy of cyclopropyl cations (11 and 13) which ring open to their allyl isomers (12 and 14) (34).
What of the Halphen reaction: the red (or pink) flag which led researchers to the CPFAs? The nroducts of the Halohen test are now considered to consist of three types of compounds bv analoev to the reaction nroducts of the model comuound
24
S-S-S
An interesting controversy arose over the reported observation (23) of products of the type 15 and 16.
Rinehart and co-workers (34) were not inclined to support the formation of 15 and 16 on the basis of their analysis of the polymer's infrared spectrum as well as the absence of methyl ketones expected following periodate-permanganate oxidation. However, Kircber demonstrated that the olefinic linkages in 15 and 16 are not degraded under these conditions, hut that ozonolysis of the polymer produced products consistent with their formation (analogous products were also produced in the reaction of sterculic acid with acetic acid) (35). In order to rationalize the formation of these products, Kircher postulated protonation of the cyclopropene ring a t its 3-position followed by ring opening to vinyl cations (11).
.. 19
I1
However, in light of the fact that structure 20 is calculated (37), using the 6-31G* basis set, to be 3.4 kcallmole less stahle than 21, and our 4-31G calculations (38)[which mimic the 6-31G* results for the comparison of 20 and 21 (37)] predict that 22 is 9.9 kcallmol less stable than 23, we offer an alternative mechanism. One need only recall that vinyl carhenes 540
Journal of Chemical Education
Compound 27 is an example of a fascinating class of compounds called tritbiapentalenes or thiothiophenes which can be described as resonance hybrids 28a and 28b (divalent sulfur) as well as 28c (tetravalent sulfur) (45). A standardized and simplified Halphen procedure has been published and corroborated by twelve laboratories (46).
The enhanced reactivity of cyclopropene toward olefinic addition reactions has been employed to implicate this unit in the Halphen test (i.e., prior reaction of the double bond inactivates CPFAs to this test). I t has also formed the basis for other quantitative assays (3). A reaction that has sparked considerable debate is the rapid addition of methyl mercaptan and other compounds containing sulfhydryl groups (-SH). This has suggested to some researchers (4,47-49) the possibility that the biological activities of sterculic and malvalic acids lie in their presumed abilities to react with sulfhydryl groups on structural proteins and enzymes. [It has, however, been noted that 1,2-dialkylcyclopropenesin benzene do not react with methyl mercaptan under an inert atmosphere over the course of several days, while reaction is rapid in the presence of oxygen & ? ) . IWe will shortly return to a discussion of mechanisms of fatty acid desaturase inhihition.
Biosynthesis and Metabolism (6) It is reasonable to suspect that, by analogy to organic synthesis, cyclopropanoid and cyclopropenoid fatty acids are made via addition of a methylene unit to an unsaturated linkage on a straight chain precursor. In fact, the source of the ring in a variety of cyclopropanoid fatty acids is known to be the amino acid methionine activated in the form of its S adenoylate (50). Thus, sterculic acid should arise from a suitable CIS-fattyacid. However, odd-carbon fatty acids are relatively rare and analogous biosynthesis of malvalic acid would require a C17precursor. A study of the incubation of newly germinated seedlings of Hibiscus syriacus ("Rose of Sharon") with l-'4C-acetate yielded the labelling pattern depicted in (V) (51).
The labelling pattern in the continuous CISchain of sterculic acid is tvoical of even-carbon fattv acids such as stearic acid. The lahiiling pattern in mal\,alioacid is identical except for t hc abstncr of labrl at C I .This itronelv s~larestsalnha oxir (52) dation as the source of malvalic acid (Vj. ~ G t h estidy with Hibiscus seedlings, employing CH3-'"C-methionine, indicated that this amino acid was indeed the source of the 9,lO-methylene group in sterculic acid and dihydrosterculic acid (V). These conclusions were supported by later work (53) which also put to rest an hypothesis (51) that stearolic acid (3) was a precursor to sterculic acid. Interestingly, traces of stearolic acid do occur naturally (54) but it is not yet clear whether they have any connection with sterculic acid metabolism. The natural occurrence of D-2-hydroxysterculicacid (55)mav also be taken as an indication of the aloha oxidation pathway (V). We note. a t this . ooint.. that svnthesis of CPFAs from cvclopropane'precursors in plants appears to he quite favorage enereeticallv desoite the 26 kcallmole increase in strain enerw incurred. he overall reaction for desaturation of a fatty aGd is given in eqn. 1 (56): Stearoyl-CoA + NADPH + H+
-
+ O2
Oleyl-CoA + NADPt
+ 2Hz0
(1)
The standard potential for this reaction can he estimated
using eqns. 2-4 (half-cell potentials are ohtained from Table 18-1 in reference 56; succinate:fumarate is employed as a model for stearoyl-CoA:oleyl-CoA). The net standard potential (Eo' = +0.523 v), obtained from summation of these equations can be NADPH + H+ t NADP+ + 2Ht + 2e- Eo' = -0.324 v (2) O2+ 4Ht + 4 e- t 2H20 En' = +0.816 v (3) succinate t fumarate + 2Ht + 2e- En' = t0.031 v (4) converted to a standard free energy using eqn. 5. If one equates the difference in strain energy of AGO = -nEoF = -4(+0.523)(23.06) = -48 kcallmole (5) cyclopropene and cyclopropane to a free energy increment (i.e., ignore the entropy difference), then the desaturation reaction AGO is about -12 kcal/mole. The metabolites of cyclopropenoid fatty acids retain the three-membered ring in the form of cyclopropane derivatives which are eliminated in urine and feces (57). This explains the absence of expired 14C02where [9,10-methylene-'4C] sterculic acid was administered to rats (58,59). Similar studies with chickens and trout found very small amounts of expired '%Or (60,61). lnhlbition of Stearic Acid Desaturation The previously noted effects of CPFAs fed to farm animals which include pasty egg yolks, hard lard, and sticky butter have their origin in increased tissue concentrations of stearic acid a t the expense of oleic acid. I n uiuo studies published in 1964 indicated that this was due to inhibition of fatty acid Ag-desaturase activity (47). Earlier, evidence had appeared suggesting that this enzyme had an active sulf'hydryl group (627, and the observation that sterculicacid reacts rapidly with methyl mercaptan and P-mercaptopropionic acid (4) strongly suggested that sterculic acid reacted irreversibly with a crucial sulfhydryl group thus deactivating the enzyme. In uiuo studies also indicated that CPFAs inhibit conversion of stearic to oleic acid (63). Further studies showed that CPFAs oroduced inhihition of desaturase art kit? u,hich c o ~ ~not l d 01: n\.trsed by addition of 2-mtwi~ptwthanol(64). Since the free sulfliydryl content ~,f'desaturase\drrcrrn~nrds~~ectruphotr~rnetr~caIIyl drcreased follou,ing addition ofCPF.\ (similar findings u,en. ohtained for L-cyscine and glutathione), it was again felt that CPFAs deactivate the enzyme by reaction with sulfhydryl (64). An in uitro kinetic study of the stearic acid desaturase system of hen livers indicated that sterculic acid is a specific and irreversible inhibitor (65).However, the fact that addition of glutathione in quantities approximately equimolar to sterculic acid did not affect the inhibition of stearic acid desaturation led one arouo .. . of researchers to conclude that st~wulicacidIS nut ugt~nrralsulthydryl en7yme inhihitor (6Gj. In 1970, I'nnde and Mend ouhl~cheddata u,hic.h challenrrd prevalent views of CPFA modes of action (67). They concluded that sterculate inhibits the desaturate system by a non-specific effect having its origin in the detergent nature of fatty acids (they observed inhibition by other fatty acids a t comparable concentrations). In further support of this view, these authors also noted the inability of added glutathione to lessen inhibition. Additionally, use of Ellman's reagent indicated that preliminary incubation with sterculate did not decrease the free sulf'hydryl content of reduced glutathione or the liver microsomal enzyme preparation. Thus, they concluded that inhibition was not the result of addition of sulfhydryl group to cyclopropene (67). These conclusions were soon challenged, in part, on the grounds that the sterculate concentrations employed were considerably higher than other groups found necessary for the near-complete inhibition of desaturase attivity (476). I t was shown that nonspecific detercent action mieht be a factor at verv. high .. concentrations. I~utat the lower c~mcentrntionsnert,;sary to produce near rornplctr desaturase inhihition (0.1 m M I , selecti\fity was ohVolume 59 Number 7 July 1982
541
served for sterculate (47b). The specificity of CPFAs for inhibition of desaturase was further explored by syntheses of two non-naturally occurring homologues of sterculic acid (68). 2-Octyl-l-cyclopropene-l-octanoic acid (sterculic acid) and its synthetic homologue 2-octyl-l-cyclopropene-l-nonanoic acid were equally effective in inhibiting stearic acid desaturation and more active than 2-octyl-l-cyclopropene-l-heptanoic acid (malvalic acid). 2-Octyl-l-cyclopropene-l-decanoic acid was not an effective inhibitor. The common feature oi the effective i n h i h i t o r s is an olrthic cyrlupropenoid carhon at C9 or ('lo thus arhievinr i o n i e s t r u r t u a l conyruence with oleic acid (68).Thus, the specificity of sterculic&id and certain closelv related cyclopropenoid fatty acids would appear . . . to be established. More recent work (69,70)on hen liver desaturase indicates that it is indeed a sulfhydryl enzyme although it is not clear whether this moup is the binding site or the catalytic site (71). This again would strongly suggest direct addition of the essential sulfhydryl group across the double bond of sterculic acid. However, addition of labelled sterculic acid to activated rat liver protein produced significant desaturase inhibition, hut solvent extraction largely freed the protein of the label (72). Thus, the association of desaturase with sterculic acid is apparently noncovalent (see also ref. 67). Jeffcoat and Pollard feel that the increased binding of sterculoyl-CoA, the annarent active form of the CPFA. over stearovl-CoA is due to either better steric fit or operatibn of a polarization mechanism. With regard to the latter effect. we remind the reader of the anomalo&ly high dipole moment of cyclopropene noted in the introduction. Miscellaneous Biological Effects T h e a l t r r t i t i o n of the transport properties of membranes by CPFAs is an extremely complex problem not wrll understood nresentlv 173. 74 I. These romouunds also are associated ~~-~~ , with other novel and Significant hfological effects. It has already been noted that cottonseed meal is an important protein source for commercial trout. These animals are highly sensitive to the carcinogenic effects of aflatoxins present in their diets (75). Sterculate has been shown to be co-carcinogenic with certain aflatoxins (76.77). thus ex~laininrthe increased hepatomas found in trbut fed'a cotton seed meal diet compared to those fed a control diet (75,78) [CPFA has also been shuwn to be carcinogenic to rainbow trout (79)].This effect is associated with auementation of DNA svnthesis (measured by increased incorporation of Wthymidine) and enhanced mitoeenesis (microsco~iccount of fraction of cells undergoing mitosis) in the hepatbcytes of trout as well as rats (80j. 1; terestinalv. while dihvdrosterculic acid was shown to be inactive, & ring-opened products of reaction of methyl sterculate with concentrated aqueous hvdrochloric acid were as a c t i v e in stimulating mitosis as methyl s t e r c u l a t t ~(XI,. The eftects of d i v t n r y CPFAs on the mixed f u n c t i o n oxidase system of r i ~ i n h o wtrout have heen studied and the induced changes employed to suggest a mechanism for co-can:inogenesis 1811. Thr significant decrease in cvtochrume P - 4 3 t 1 content o b servedi82) might be in some way related to the ability of this nrotein to react. under reducine conditions, with carhenes (83, which might conceivably i;e g e n e r a t t ! d from CI'12As. One additional novel effect is that sterculic arid a r w a r e n t l v has a moderate ahility to inhibit lepidopterous larv-aigrowth (85). This effect would he most significant in young seeds where concentrations are highest and which are, in fact, the ooint of entry of certain larvae (85). Thus, a subtle plant ~elf-protecti~n mechanism is suggested. Although there exist numerous pathways for transfer of CPFA to man through commercial food, one author, at least, feels that potential dangers are small. Scarpelli notes that the levels of CPFA are very low and that cooking probably destroys the activity of remaining material (80). Finally we note the recent discovery of the first known cyclopropenone derivatives to be found in nature (86).
Acknowledgment We wish to thank the American Chemical Society for summer support of J. Harris, Plainfield High School, as a Project SEED researcher.
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542
Journal of Chemical Education
6t \Imr 1I.H K .ndspmmr.F . n " B w h e m ~ . t nnlPlnm."\'x 4 x.mvf.1' ti o d I' I n . E E h.'o.i.r3 i.A;drm#. P M \ $ hru York. 19RO.yp W i n , ? \.2,",.
(13) (141 (15) (16)
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