Phytochemlstry,1973.Vol. 12, pp. 2759to 2764. Pergamon Press. Printed IIIEngland.

CAROTENE EPOXIDES OF L YCOPERSICON ESCULEh’TUil4

AVRAHAM BEN-AZIZ*, GEORGE BRITTON and TREVOR W. GOODWIN
Department of Biochemistry, University of Liverpool, Liverpool L69 3BX

(Received 8 May 1973. Accepted 1 June 1973)

Key Word Index-Lycopersicon esculentum; Solanaceae; tomato; carotenoids; epoxides; apo-lycopenals.

Abstract-A series of oxygenated carotenoids has been isolated from tomatoes. Two of these compounds
have been identified, by comparison of their chromatographic and spectroscopic properties with those of
semisynthetic samples, as epoxides of lycopene (1,2_epoxy-1,2-dihydro-$&carotene and 5,6-epoxy-5,6-
dlhydro-#,$-carotene). The other related compounds have been identified by their chromatographic,
spectroscopic and chemical propertles as mutatochrome (5,8-epoxy-5,8-dihydro-&/l-carotene) and epoxides
of phytoene (1,2-epoxy-1,2,7,8,11,12,7’,8’,11’,12’-decahydro-~,tgcarotene), phytofluene (1,2epoxy-1 >2 97 ,89
11,12,7’,8’-octahydro-#,+uotene and 1,2-epoxy-l,2,7,8,7’,8’,11’,12’-octahydro-~,~~arotene) and {-
carotene (l,Zepoxy-1,2,7,8,7’,8’-hexahydro-~,9-carotene). The presence in tomatoes of apo-6’-lycopenal
(6’-apo-+caroten-6’-al), 8’-apo-lycopenal (8’-apo-&caroten-8’-al) and lycoxanthin ($,+caroten-16-01) has
been confirmed by comparison with authentic samples.

INTRODUCTION
THE CAROTENOID hydrocarbons of several strains of tomato have been well characterized,1-3
but no recent systematic work on the xanthophylls of tomatoes has been reported.4 Kuhn
and Grundmann’ reported that the xanthophyll fraction of normal tomatoes comprised
mainly lutein @,<-carotene-3,3’-diol) and zeaxanthin @,/3-carotene-3,3’-diol) and Curl6 used
counter-current distribution to demonstrate the presence of a large number of xanthophylls,
including the normal chloroplast xanthophylls. Curl6 also detected lycoxanthin (I,$#-
caroten-16-01, X) in the mono1 fraction in agreement with the earlier work of Zechmeister
and von Cholnoky.7 Zechmeister and Pinckard* isolated a compound that was more polar
than phytofluene but had a similar absorption spectrum. They concluded that this was a
monohydroxyphytofluene which they named phytofluenol.
Recently we reported9 the characterization of a compound that was identified from its
chromatographic and spectroscopic properties as phytoene epoxide (1,2-epoxy-1,2,7,8,11,
12,7’,8’,11’,12’-decahydro-#,$-carotene, I), and noted the presence in tomatoes of other
related compounds with similar chromatographic polarity. We now report the characteriza-
tion of several of these compounds.
* Present address: Agricultural Research Organization, Pestlclde Chemistry and Residues Research
Laboratory, Volcani Research Centre, P.O.B. 6, Bet-Dagan, Israel.
1 WILLIAMS,R. J. H., BRITTON,G., CHARLTON,J. M. and GOODWIN,T. W. (1967) Biochem. J. 104,767.
2 RAYMUNDO,L. C., GRIFFITHS,A. E. and SIMPSON, K. L. (1967) Phytochemistry 6, 1527.
J RAYMUNDO,L. C., GRIFFITHS,A. E. and SIMPSON,K. L. (1970) Phytochemistry 9, 1239.
4 EDWARDS,R. A. and REUTER,F. H. (1965) J. Food Sci. Technol. 2, 66.
5 KWN, R. and GRUNDMANN,C. (1932) Chem. Ber. 65, 1880.
6 CURL, A. L. (1961) J. Food Sci. 26, 106.
’ ZECHMEISTER, L. and VON CHOLNOKY, L. (1936) Chem. Ber. 69,422.
8 ZECHMEISTER, L. and PINCKARD,J. H. (1948) Experientiu 4, 471.
g BRITTON,G. and GOODWIN,T. W. (1969) Phytochemistry 8,2257.
2759
2760 A. BEN-AZIZ, G. BRITTON,and T. W. GOODWIN

RESULTS

Chromatographic examination of the unsaponifiable material from an acetone extract of

tomatoes showed the presence of a large number of oxygenated carotenoids. A group of
pigments with polarity intermedlate between the hydrocarbons and the monohydroxy-
carotenoids could, however, be isolated, and several of the individual compounds in this
group have been purified and characterized.

Carotene Epoxides
Included in this fraction was a series of compounds with polarity similar to that of
squalene-2,3-epoxide and mutatochrome (5,8-epoxy-5,8-dihydro-,B&carotene, VII). These
compounds were separated by successive TLC in three systems to give a series similar to the
series of hydrocarbons present in the extract. The compounds could not be saponified, did
not form acetates or trimethylsilyl ethers, and their absorption spectra were not changed by
NaBH, or dilute acid. This shows the absence of ester, hydroxyl, carbonjll and cyclic
5,6-epoxide groups. The pigments were characterized by their chromatographlc properties,
absorption spectrum and MS. Insufficient material was avallable (ca. 100 pg of each
pigment) for confirmation of the proposed epoxide structures by NMR spectroscopy,
but two of the compounds were shown to be identical (chromatographic properties,
absorption and mass spectra) with two epoxides of lycopene produced by treatment
of lycopene with m-chloroperbenzoic acid.

Phytoene Epoxide (1,2-epoxy-1,2,7,8,11,12,7’,8’,11’,12’-decahydro-~,f~-carotene, I)

The characterization of this compound has been reported previously.’

Phytofluene Epoxides (1,2-epoxy-l,2,7,8,7’,8’,11’,12’-octahydro-~,+carotene, II, and 1,2-

epoxy-1,2,7,8,11,12,7’,8’-octahydro+&carotene, III)
Closely associated with the phytoene epoxide, but separable from it by TLC on MgO-
Kieselgur was a fraction whose bright greenish-white fluorescence under UV light, and UV
absorption spectrum (X,,, in petrol. at 331, 348, 367 nm) indicated it to be a phytofluene
derivative. The mass spectrum showed the parent ion, M+ to be at m/e 558 (50 %, C,,H,,O),
but no fragment ion was present at m/e 540 (M-H,O), thus confirming the absence of
hydroxyl groups. [Fragment ions due to loss of water are observed in the mass spectra of
hydroxyphytofluenes from Rhodospirillum rubrum’O (I ,2,7,8,11,12,7’,8’-octahydro-#,#-
caroten-l-01) and berries of Solanum dulcamara” (probably 7,8,11,12,7’,8’-hexahydro-#,#-
caroten-16-ol).] Major fragment ions were observed at m/e 353 (16 %, M-205, metastable
at m/e 223; 353’1558 = 223.3) and 405 (5x, M-153, metastable at m/e 294; 4052/558 =
294.0) due to cleavage of the ‘bis-allyhc C-l 1’,12’ and C-7,8 bonds respectively.’ 2 This is
consistent with structure II, 1,2-epoxy-l,2,7,8,7’,8’,11’,12’-octahydro-~,~-carotene. The MS
also contained strong fragment ions at m/e 421 (2.5 %, M-137, metastable at m/e 318;
421’/558 = 317*6) and 337 (8x, M-221, metastable at m/e 203.5; 3372/558 = 203.5) as
expected for cleavage of the C-7’,8’ and C-l I,12 bonds of the isomeric phytofluene epoxlde,
1,2-epoxy-l,2,7,8,11,12,7’,8’-octahydro-~,+carotene (III). The ratios of the intensities of the
353 and 405 to the 337 and 421 ions indicated that II and III were present in a ratlo of ca.
2: 1.
lo MALHOTRA,H. C., BRITTON,G. and GOODWIN,T. W. (1970) FEBS Letters 6, 334.
I’ DUNKEYSON,S. F., BRITTON,G. and GOODWIN,T. W. unpubhshed results.
I2 WEEDON,B. C. L. (1969) Fortsch. Chem. Org. Naturst. 27, 81.
 Carotene epoxides of Lycopersicon esculentum 2761

[-Carotene Epoxide (l,Zepoxy-1,2,7,8,7’,8’-hexahydro-$,$-carotene, IV)

This compound, closeIy reIated to I, II and III, had X,,, (petrol.) at 377, 400, 425 nm
indicative of the conjugated heptaene chromophore of a I-carotene derivative. The mass
spectrum had the parent ion M+ at m/e 556 (74’?, C4,,Hs,,0) and major fragment ions at
m/e 419 (4x, M-137, metastable at m/e 316; 419’/556 = 3158) and 403 (4.5%, M-153,
metastable at m/e 292; 4032/556 = 292.1) due to cleavage of the bis-allylic C-7’,8’ and
C-7,8 bonds respectively. No fragmentations involving the loss of water were detected. The
experimental data are consistent with structure IV, 1,2-epoxy-1,2,7,8,7’,8’-hexahydro-#,+
carotene.

Lycopene-1 ,Zepoxide (1,2-epoxy-1,2-dihydro-#,$-carotene, V)

A further compound was obtained which had an absorption spectrum similar to that of
lycopene, with Amax(petrol.) at 443, 469, 500 nm. The MS showed the molecular ion M+
at m/e 552 (100x, C40H560) and had a major fragment ion at m/e 483 (2%, M-69, meta-
stable at m/e 423; 4832/552 = 422.6) and a less intense ion at m/e 467 (lx, M-85) due to
cleavage of the C-3’,4’ and C-3,4 bonds respectively. Fragment ions were also observed at
m/e 473 (I%, M-79), 460 (6 %, M-92, metastable at m/e 383, 4602/552 = 383*3), 446 (28 %,
M-106) and 394 (2 %, M-l 58) due to fragmentation of the polyene chain,12*13 and multiple
losses of 69 or 85 m.u. from these fragments were also observed, especially at m/e 377 (2 %,
M-106-69, metastable at m/e 319; 3772/446 = 318.7). No fragmentations involving loss of
water were detected. This compound was therefore assigned structure V, lycopene-1,Z
epoxide (1,2-epoxy-1,2-dihydro-#,$-carotene). The absorption spectrum and MS were
identical to those of a sample of lycopene-1,Zepoxide obtained by reaction of lyco-
pene with m-chloroperbenzoic acid, and the tomato compound and the semi-synthetic
lycopene epoxide were not separated by TLC on silica gel G with 10% ether in petrol.,
or on MgO-Kieselgur G (1: 1) with acetone-benzene-petrol. (2 : 2 : 1) as developing solvent.

Lycopene-5,6-epoxide (5,6-epoxy-5,6-dihydro-$,+-carotene, VI)

Isomerlc with the above (V) (MW552, C40H560) but separable from it by TLC on
MgO-Kieselgur G was a compound with absorption spectrum (X,,, in petrol. at 431, 454,
l3 VETTER,W., ENGLERT,G., RIGASSI,N. and SCHWIETER,
U. (1971) in Curotenoids (ISLER,O., ed.), p. 189,
Birkhluser, Base].
2762 A. BEN-AZIZ, G. BRITTON, and T. W. GOODWIN

483 nm) indicative of an acyclic conjugated decaene chromophore. The mass spectrum,
which had the parent ion M+ at m/e 552 (80%, C,,H,,O) again showed no losses of water,
but did contain fragment ions at m/e 473 (I%, metastable at m/e 405; 4732/552 = 405.3),
460 (11 %, metastable at nlje 383; 4602/552 = 383.3), 446 (36 %) and 394 (2%) due to losses
of 79, 92, 106 and 158 m.u. respectively by rearrangement and cleavage of the polyene
chain, and at m/e 483 (2*5x, M-69, metastable at m/e 423; 4832/552 = 422.6), 391 (I %,
M-92-69, metastable at m/e 332; 3912/460 = 332.4) and 377 (3 %, M-106-69, metastable at
m/e 319; 3772/446 = 318.7) due to cleavage of the C-3,4 and C-3’,4’ bonds. No losses
of 85 m.u. were observed. From the available data, this compound was tentatively
identified as lycopene-5,6-epoxide (5,6-epoxy-5,6-dihydro-#&carotene, VI) and this was
confirmed by comparison with lycopene-5,6-epoxide obtained as a major product of
the reaction of lycopene with nz-chloroperbenzoic acid. The absorption spectrum and MS
of the two samples were identical, and the two samples were not separated by TLC on
silica gel G with 10 “/, ether m petrol. or MgO-Kieselgur G (I: 1) with acetone-benzene-
petrol. (2: 2: 1) as solvent.

Mutatochrome (5,8-epoxy-5,8-dihydro-&3-carotene, VII)

A compound was also isolated which had an absorption spectrum (A,,, in petrol. at 404,
427, 452 nm) and chromatographic properties identical to those of mutatochrome (5,8-
epoxy-5,8-dihydro-/3,/3-carotene). The MS was very similar to that shown by Budzikiewicz
et a1.,14 and had the parent ion, M+ at m/e 552 (68 %, C,,H,,O) and fragment ions at m/e
460 (lx, M-92, metastable at m/e 383; 4602/552 = 383.3) and 446 (lx, M-106) and
especially at m/e 472 (36x, M-80, metastable at m/e 404; 4722/552 = 403.6), 205 (36%)
and 165 (35 %) indicative of the furanoid oxide structure.12*1s This compound was therefore
identified as mutatochrome, 5,8-epoxy-5,8-dihydro-@-carotene (VII).

Other Carotenoids
Three other acyclic carotenoids were also isolated from this chromatographic fraction.
Two of these, which were similar in polarity to the epoxide series and had absorption spectra
lacking in fine structure, typical of carotenoids containing a carbonyl group in conjugation
with the polyene chromophore, were identified as apo-lycopenals. The third compound,
which was more polar, was identified as lycoxanthin.

Apo-6’-lycopenal(6’-apo-+-caroten-6’-al, VIII)
The absorption spectrum of this compound (A,,,,, in ethanol at ca. 475 nm) indicated the
presence of a carbonyl group in conjugation with a decaene system, and this was confirmed
by a shift m the absorption maxima to 430, 455, 485 nm on reduction with NaBH,. The
spectrum of the reduced product indicated a decaene chromophore and showed fine structure
typical of an acyclic carotenoid. The MS of the natural carbonyl compound had the parent
ion, M+ at m/e 442 (100 “/,, C,,H,,O) and a strong fragment ion at nrje 373 (7.5 “/,, M-69,
metastable at m/e 315; 3732/442 = 314.8) due to cleavage of the ‘bis-allyhc C-3,4 bond.
The spectroscopic data were consistent with the formulation of this compound as apo-6’-
lycopenal (6’-apo-JI-caroten-6’-al, VIII). The absorption spectrum and MS were identical
to those of a sample of apo-6’-lycopenal obtained by KMnO, oxidation of lycopene, and

I4 BUDZIKIEWICZ, H.,
BRZEZINKA, H. and JOHANNES,B. (1970) Monatsch. Chem. 101,579.
” BALDAS J PORTER Q. N., VON CHOLNOKY, L , SZABOLCS,J. and WEEDON, B. C. L. (1966) Chem. Commun.
852. ’ .’ ’
 Carotene epoxides of Lycopersicon esculentum 2763

the identity was confirmed by co-chromatography; the two samples could not be separated
by TLC on silica gel G with 10 % ether in petrol. or on MgO-Kieselgur G (1: 1) with acetone-
benzene-petrol. (2: 2: 1) as solvent. The presence of apo-6’-lycopenal in extracts of
tomatoes has previously been reported by Winterstein et a1.16

Apo-8’-lycopenal(8’-apo-+caroten-8’-al, IX)
Closely associated with the above aldehyde (VIII) was a compound which had X,,,
(ethanol) at cu. 465 nm and which, on NaBH, reduction gave a product with well-defined
h,,, (ethanol) at 416,441,465 nm, indicating an acyclic nonaene chromophore, and showing
that one carbonyl group had been reduced. The MS had the molecular ion, M+ at m/e 416
(100x, C30H400) and a fragment ion at m/e 347 (9 %, M-69, metastable at m/e 289;
347’/416 = 289.4) due to cleavage of the C-3,4 bond. From the available data, this
compound was identified as apo-8’-lycopenal (8’-apo-$-caroten-8’-al, IX), the presence of
which in extracts of tomatoes has also been reported previously.16 The identification was
confirmed by comparison of the absorption spectrum and MS with those of a sample
of apo-8’-lycopenal obtained by KMnO, oxidation of lycopene, and by co-chromatography
of the two samples, which were not separated by TLC on silica gel G with 10% ether in
petrol. or on MgO-Kieselgur G (1: 1) with acetone-benzene-petrol. (2: 2: 1) as solvent.

Lycoxanthin (#,#-caroten-16-01, X)
Small amounts were isolated of a compound with an absorption spectrum (&,,,x in
ethanol at 445,470, 502 nm) identical to that of lycopene but with the polarity of a mono-
hydroxycarotenoid. The MS had the parent ion, M+ at mJe 552 (100x, Cd0Hs60), major
fragment ions at m/e 534 (32x, M-H20, metastable at m/e 517; 5342/552 = 516*6), 483
(lo%, M-69, metastable at m/e 423; 4832/552 = 422.6) and 467 (5x, M-85), and fragment
ions due to multiple losses of the above fragments with toluene (92 m.u.) and xylene (106
m.u.). The details of the MS were very similar to those quoted by Markham and Liaaen-
Jensen,” and illustrated by Enzell et al. I8 The absorption spectrum and MS were identical
to those of lycoxanthin isolated from Solanum dulcamara fruits,” and the two samples
were not separated by TLC on silica gel G with 40% ether in petrol. or on MgO-
Kieselgur G with acetone-benzene-petrol. (2:2: 1) as solvent. This compound was
therefore lycoxanthin ($,+caroten-16-01, X) the presence of which in tomatoes has
previously been reported.6*7
DISCUSSION
Tomato fruit contain a very large number of oxygenated carotenoids, most of which are
present in very small amounts. Several of these compounds have been isolated and
characterized by their chromatographic, spectroscopic and chemical properties as epoxides
of the normal tomato acyclic carotenoid hydrocarbons phytoene, phytofluene, I-carotene
and lycopene. The epoxide nature of these compounds was established by comparison of
the lycopene derivatives with semi-synthetic epoxides prepared from lycopene. Although
naturally occurring epoxides of cyclic carotenes are well known,19*20 this is the first report
of the occurrence of epoxides of acyclic carotenes in extracts of natural tissues. Members of
I6 WINTERSTEIN,A., STUDER,A. and R~~EGo, R. (1960) Chem. Ber. 93,295l.
I7 MARKHAM, M. C. and LIAAEN~ENSEN,S. (1968) Phytochemrstry 7, 839.
I8 ENZELL, C. R., FRANCIS, G. W. and LIAAEN-JENSEN, S. (1969) Acta Chem. Stand. 23,727.
I9 KARRER, P. and JUCKER,E. (1950) Carotenoids (BRAUDE, E. A., translat.), Elsevler, New York.
2o WEEDON,B. C. L. (1971) in Carotenoids (ISLER, O., ed.), p. 29, BlrkhBuser, Basel.
2764 A. BEN-AZIZ, G. BRITTON,and T. W. GOODWIN

this new series of carotenoid epoxides have also been detected in fruit of Woody NIghtshade
(Solanum dulcamara). 1 l
The biological significance of the occurrence of these compounds is not known. They are
present in very small quantities (ca. 100 pg isolated from 900 g frmt), but do not appear to
be artefacts of the isolation and purification procedure. 21 It is possible that these epoxides
of acyclic carotenoids may be formed by the fruit in response to injury, in a similar manner
to the rapid formation of p-carotene epoxldes m excised leaves. 22 It IS also possible that these
epoxides and the apo-lycopenals may be early products of the oxidative degradation of the
carotenes m the senescing tlssues,23 since a prehminary investigation indicates that these
compounds may be present in larger quantities In over-ripe tomatoes.21

EXPERIMENTAL
Materials. Tomatoes (900 g) were bought from a local shop. Lycopene-1,2-epoxlde and lycopene-5,6-
epoxlde, prepared by treatment of lycopene with m-chloroperbenzolc acrd, were kmdly provided by Miss
M. K. McLaurin. Apo-6’-lycopenal and apo-8’-lycopenal, obtained by KMnO, oxldatlon of lycopene were
kmdly provtded by Mr. D. J. Brown. Lycoxanthm, Isolated from Solanunr dulcamuru fruit, was the kmd gift
of Mr. S F. Dunkeyson. Petrol (hght petrol , b p. 40-60”), C6Hs and Et,0 (dlethyl ether) were dried over
sodium-lead alloy, and acetone for chromatography was drted over K2C03. All solvents were dlstdled before
use, ether from reduced Iron powder.
Extractron andpurificatzon methods. LIpId material was extracted with acetone and sapomfied by standard
procedures.“’ The unsapomfiable material was chromatographed on a column of neutral alumina (100 g,
actlvlty grade III). Hydrocarbons were eluted with 20% C,H, m petrol, and polar substances then removed
with 5 ‘A EtOH m Et,O. This polar fraction was rechromatographed on a second column of neutral alumina
(40 g, actlvlty grade III). Traces of lycopene were removed wrth 20% C6Hs m petrol, a second fraction was
eluted with 10% Et20 m petrol, and the remammg polar material was eluted with 5% EtOH m EtzO.
The second (10% Et,0 m petrol) column fraction was chromatographed on thm layers of silica gel G m
10% ether m petrol. This gave several bands, three of which were removed and eluted. Band 1 (R, 0.7) was
pale yellow, and fluoresced greenish-white under UV hght, whereas bands 2 (R, 0 5) and 3 (Rf 0 35) were
red Mutatochrome and the epoxides of phytoene, phytofluene and c-carotene were obtained from band 1
as previously described,g and were further purified by TLC on slhca gel G with 5 yd Ft,O m petrol before
determmation of their MS
TLC of band 2 on MgO-Kleselgur G (1: 1) m acetone-C6Hs-petrol. (2:2: 1) gave lycopene-1,2-epoxide
(R, 0 4) and lycopene-5,6-epoxlde (R, 0.6), and these were further purified by TLC on sdlca gel G with 8 %
Et*0 m petrol.
TLC of band 3 on MgO-Kieselgur G (1.1) with acetone-&H,-petrol (2: 2: 1) gave two mam products,
apo-6’-lycopenal (RI 0.3) and apo-8’-lycopenal (Rf 0.45), which were further purified by TLC on slhca gel G
with 10 % Et,0 m petrol.
The third (5 % EtOH m EtzO) column fraction was chromatographed on thm layers of sdrca gel G with
Et,O-C,H,-petrol. (9: 1 :lO). Many bands were observed, but a red band (Rf 0 7) was removed, eluted and
rechromatographed in the same system. This red band was then chromatographed on a thin layer of MgO-
Kieselgur G (1’1) with acetone-C,Hs-petrol (2:2:1), to give Iycoxanthm (RI 0 3), which was further
purified by TLC on slhca gel G with 40% Et20 m petrol.
Co-chromatography. Lycopene epoxldes and apo-lycopenals were co-chromatographed with semIsynthetIc
samples, and lycoxanthrn with an authentic natural sample, on slhca gel G and MgO-Kleselgur G (1: 1) m
the solvent systems described in the Results.
C/zernzca/ reactrons. Attempted acetylatlon, sllylatlon, acid lsomerlzatlon and NaBH, reduction were
performed by standard procedures.*“
Spectra Absorption spectra of the epoxldes were determined m petrol and of lycoxanthm and the apo-
lycopenals in EtOH. MS were determmed by Mrs. A M. Ball and Mr J. R. Ireland, in an A.E.I. MS12
instrument, with the direct msertlon probe, and at an ion source temp. of 210.-350”.
Acknowledgements-We thank the Science Research Councd for financial support. A.B.-A. thanks the
Hebrew University of Jerusalem for a research fellowshlp, and the Umverslty of LIverpool for the award of
a Johnston FellowshIp m Blochemlstry.
” BEN-AZIZ, A. unpublished results.
” GLOVER,J. and REDFEARN,E. R. (1953) Blochem. J. 54, VIII.
23 CHICHESTER,C. 0. and NAKAYAMA,T. 0. M. (1965) in Chemrstry arzd Biochemutry of Plant Prgments
(GOODWIN,T. W., ed.), p. 439, Academic Press, London.
24 BRITTON,G. and GOODWIN,T. W. (1971) Meth Enzymol. 18C, 654.