Recent progress in carotenoid chemistry

RECENT

PROGRESS

IN

CAROTENOID

CHEMISTRY SYNNOVE LIAAEN-.]ENSEN a n d ARNE JENSEN

Institute of Organic Chemistryand Norwegian Inst. of Seaweed Research, The Technical Univ. of Norway, Trondheim I. INTRODUCTION

THE isoprenoid compounds named carotenoids by Tswett in 1911, which are so familiar to layman and scholar because of their presence in gaily-coloured flower heads, in red and yellow vegetables and fruits, vividly tinted autumn leaves and in the feathers or skin of many birds and Crustaceae, comprise some 150 different chemical entities. Formally the carotenoids may be regarded as derived from one parent substance, namely the widely distributed, aliphatic carotene, lycopene.

~, ,-,

~,,

CH3

n 3~'-~ /~n3H

[

CH 3

H

I

H~C--C /

H

H

H

H

H

I~

tq2 C

~'CH 2

[

Hc&Cc~C~c.C%~C%~C~c~C%~C%~C%.C%~C~c.CH ~CH _ L

II

H

Mz('~.c/C--C H3 Hz

H

H

H

H

]

CH 5

H

I

CH 3

H H:C/

~'CH3

Lycopene (complete formula)

o

Lycopene (abridged formula)

By simple chemical reactions, such as partial hydrogenation, dehydrogenation, cyclization, occasionally including methyl migration and aromatization, introduction of oxygen functions and cleavage of the C40-molecule at various sites, all carotenoids may, theoretically, be derived from lycopene. A review of the recent progress in the field of carotenoid chemistry will naturally be restricted to the development which has taken place since the appearance of the comprehensive monograph by Karrer and Jucker in 1948.189,190 To limit the topic further, A-vitamins and related substances have been excluded from the present survey, partly because recent discussions of this field have been delivered by Mackinney24s,Islet and Zeller159and Olsonz56. Attempts have been made to avoid information which is easily available in 133

Progress in the Chemistry of Fats and other Lipids well-known monographs. 102, 352 A detailed survey of natural occurrence and distribution is thus omitted. Since new methods have been developed and perfected for the structural elucidation of carotenoids in semimicro- and micro scale, emphasis has been put on recent improvements in isolation procedures, and other analytical methods. Moreover, it was considered imperative to present the more important chemical evidence on the naturally occurring carotenoids for which reliable structural formulae have been established during the last fifteen years, particularly because no review covering this topic has appeared during the period. Recent contributions to the establishment of chemical structures for carotenoids with partly known structures have also been included. The most remarkable achievement of the period is undoubtedly the performance of an explosive development in the total synthesis of carotenoids. A compilation of the general principles applied therefore seems justified, despite the fact that full treatment of this field has appeared elsewhere. In this connection reactions useful for partial synthesis of carotenoids have also been dealt with. Biochemical aspects of carotenoid chemistry have been reviewed quite frequently during recent years. Only a summary of the function, biosynthesis and metabolism of these pigments has been included in the present survey. 1I. METHODS

A. Isolation procedures The general methods of isolation of carotenoids from plant and animal material have been discussed in some detail by Karrer and Jucker. 19° Since then certain improvements of the old techniques and some development ef new procedures have taken place. The principal problems met with are those of homogeneity and purity of the samples, degradation or other changes of pigments prior to and during extraction, and efficiency of the extraction process. The importance of securing a pure and homogeneous sample of the material to be investigated may be demonstrated by the following constructed example: Upon extraction of a lichen material low in carotenoids (20 mg/100 g of dry material127), contaminated with I per cent of Trentepolia iolitus (2-3 g of carotenoids/100 g of dry matter9S), more than haft of the pigments in the extract of this practically homogeneous lichen material would originate from the contaminating algae. As a general rule, air-drying of samples before extraction, as previously recommended for most plant materials, ag° should be avoided since loss of pigments may occur during drying. 91 When drying has to be carried out, lyophilization is the method of choice. In most cases direct extraction of the fresh material with a water miscible solvent, as soon as possible after the harvest, constitutes the superior method. Acetone 293,24~, 77 or acetone ÷ petroleum ether (for less polar carotenoids 25, 26) seems to be the most suitable solvent. 134

Recent Progress in Carotenoid Chemistry The extraction is preferably carried out by grinding the material with quartz sand in a beaker 25, 26 or by beating the material with acetone in a Waxing Blendor.25, 122 In the latter case, frequent addition of small lumps of solid carbon dioxide keeps the temperature around the freezing point and creates a protective atmosphere which secures a very gentle extraction. 172 Antioxidants such as quino126 or basic compounds like calcium carbonate 122 or, better, pyridine and dimethylaniline 3°6 may be added to the solvents to reduce oxidation and to avoid rearrangements of carotenoids by plant acids. The extracted pigments are often transferred to benzene, petroleum ether or ether, by addition of any of these solvents and water containing sodium chloride or ammonium sulphate. 26 The use of salt solutions prevents the formation of emulsions. Further washing with water (or salt solution) is necessary to remove the original solvent used for extracting the raw material. Following drying over anhydrous sodium sulphate, the extract is concentrated at reduced pressure and room temperature to a convenient volume. In many cases direct concentration of the original pigment extract at reduced pressure and below 0°C (which means freeze drying at the end) may be carried out without significant loss of carotenoids. Saponification is to be omitted when not made necessary by presence of disturbing quantities of non-carotenoid lipids, as some carotenoids are destroyed by even very weak bases. 219 If saponification is omitted it will be necessary to check for the presence of carotenoid esters at a later stage of the investigation. Before the pigments are subjected to separation into single components, grouping into an epiphasic and a hypophasic fraction may be helpful. This is accomplished by partitioning the carotenoids between petroleum ether and aqueous methanol. 259, ~oa The less polar carotenes will go to the first and the more polar xanthophylls to the latter phase. In order to minimize cis-trans isomerization of the carotenoids, all operations connected with isolation and separation of the pigments should be carried out in dim light and as rapidly as possible. There is no time to relax until the pure, crystalline pigment is stored under nitrogen in the deep-freeze. More detailed treatment of the isolation procedures of carotenes and carotenoids are found in the report published by the Society for Analytical Chemistry; 6 in the chapter on carotenoids by Goodwin in Modern Methods of Plant Analysis; 1°5 in Linsken's book on paper chromatography; 12~ in a monograph by Liaaen-Jensen, ~37 and in the review by Brubacker. 32

1. Chromatography Only in very rare cases214 will a plant or an animal material contain so few carotenoids that the components may be crystallized in a relatively pure state directly from an extract, even after saponification and partition into epi- and hypophasic fractions. To separate the rather complex mixtures of pigments 135

Progress in the Chemistry of Fats and other Lipids usually encountered, one will have to resort to various forms of chromatography or countercurrent distribution. Among the more recent advances in these fields may be mentioned the introduction of polyethylene columns2 the development of paper-chromatographic techniques,')ST, 170 and separation by thin-layer chromatography, 29s and the development of the countercurrent distribution method. 56,a9 The fact that rigidly standardized adsorbents for column and thin-layer chromatography have been made commercially available must also be regarded as a valuable improvement to the field. Column chromatography. The adaptation of column chromatography to the separation of carotenoids has been discussed in some detail by Karrer and Jucker. 190 More recent treatments of a general nature are to be found in the books of Lederer and Lederer, 21s and by Heftmann 126 and in an Annual Priestley Lecture delivered by Strain. a°9 The chromatographic systems in common use are based on either of the two different processes of adsorption and partition. To the list of useful adsorbents given by Karrer and Jucker 19° has been added the activated cellulose of Mille et al. 2~2 Strain 310 has also given a useful list of adsorbents and solvents for chromatography of carotenoids and has discussed the basis of selectivity of the possible systems. For certain very polar carotenoids a special brand of magnesium silicate has been successfully applied. 3za Generally it may be stated that non-polar carotenes are best separated on calcium hydroxide and alumina columns, while calcium carbonate and magnesium oxide seem to work best with carotenoids of intermediate polarity. Very polar pigments, like the carotenoid acids, are best separated on sugar or cellulose columns. The possiblity of separating carotenoids by partition chromatography does not seem to have been investigated very thoroughly. Purcell ~7° has developed a partition method on silica columns, and the system containing 80 per cent aqueous methanol on polyethylene columns, introduced by Anderson, Blass and Calvin, 9 is in reality a reversed phase partition system. The sequence of pigments on the column is therefore the reverse of that obtained in adsorption systems. Thin-layer chromatography. The recent development of the old chromatographic technique of Izmailov and Shraibed 6° into the modern method of thinlayer chromatography, 295, 296 has been successfully applied to the field of carotenoids by a number of authors, a3s, 3a7,156,298,320, 73, Sl, 23 The adsorbents--kieselgel, alumina and calcium hydroxide are the most widely used--are applied on glass plates in a thin layer (0.1-2 mm), whereupon drying and activation of the adsorbents follow. The samples to be tested are applied as small spots in one corner (for two-dimensional development) or along one end of the plate, and the development is usually carried out in an ascending way by standing the plate in a small glass chamber containing a suitable amount of the solvent system. The development usually takes 2060 min. The coloured spots of the carotenoids may be scraped off the plate, the 136

Recent Progress in Carotenoid Chemistry pigment eluted, and the light absorption spectrum determined. Some examples of the separation obtainable by thin-layer chromatography are given in Table 1. The method is very suitable for a rapid investigation of carotenoids in fractions obtained by column chromatography of extracts of plant and animal materials. Identification by co-chromatography on thin-layer plates does not always seem to be conclusive, but gives good presumptive evidence for identity.

Table 1. R f-values × 100 for carotenoids separated by thin-layer chromatography 297 Compound

System

System

1

a-Carotene /3-Carotene e-Carotene Lycopene Cryptoxanthin Lutein Zeaxanthin Violaxanthin

2

88 84 40-50 10-20

m

m

m

74 55 57

System 3

100 100 100 100 75 35 24 21

System 1: Calcium hydroxide-kieselgel G (6 ÷ 1); petroleum ether-benzene (98 -- 2). System 2: Calcium hydrc.xide-kieselgel G (6-~ 1); benzene-methanol (98 -4- 2). System 3 : Kieselgel G; methylene chloride-ethyl acetate (80 + 20). It should be mentioned that the method is not well suited for quantitative work since losses on the thin and heavily exposed plates are considerable, and since quantitative removal of the coloured spots from the plates is somewhat cumbersome. A number of pertinent reviews on thin-layer chromatography and the application of the method to the field of carotenoid chemistry are available.297,298,249,339,19 Paper chromatography. Ordinary paper chromatography has not yet found any wide application in the field of carotenoid chemistry, although a number of investigators have used the method with success. 165, 10,288 The development of paper-chromatographic methods for the separation of carotenoids may have been delayed by the high efficiency and broad applicability of the column chromatography within this field. However, the large number of publications reporting more or less successful attempts to develop suitable systems for the separation of carotenoids and other chloroplast pigments on paper, clearly demonstrates the need for these means. The best adsorption systems developed seem to be those based on impregnated paper116, 27 or on paper containing special types of fillers. 1~7, 173 Useful reverse phase partition systems have also been developed, e49, 307, 27 Several types of 137

Progress in the Chemistry of Fats and other Lipids

filter paper, containing special fillers, have been made commercially available* and have been found well suited to both qualitative and quantitative separation of carotenoids and chlorophylls, lv3, 16< 167,174 It has been possible to ascribe reproducible R1-values to the different carotenoids by carefully standardizing the chromatographic procedures 165, 17z (see Table 2), and the pigment recovery Table 2. R1-values qf carotenoid pigments 173 on Schleicher & Schiill fiher paper No. 287 Member Carotenoid

fl-Carotene Cryptoxanthin Zeaxanthin Lutein Violaxanthin Astaxanthin Rhodoxanthin Fucoxanthin y-Carotene Lycopene Anhydro-rhodovibrin Spirilloxanthin

Chloroxanthin Rhodopin Rhodovibrin

a-Bacterioruberin

R f-value t

of the set

0°0

2 3{

5%

lO%

20%

all-trans all-tram all-tram all-trans all-trans all-trans all-tram all-trans all-tram all-tram all-trans neo A all-tram neo a neo b all-trans all-tram all-tram neo A neo B neo U all-trans neo A neo B

0'95 0"29

0"98 0"62 0"09

0"98 0"81 0'30 0"39 0"18

0'98 0"91 0'59 0"72 0"44 0"57 0"27 0"40

0.87 0.91 0.83 0.85 0-72 0.81

0'10 0"68 0"53

0'13

0"86 0'35 0"50 0"18 0"31 0"48 0'46

0'60 0"75 0"40 0'54 0"73 0"73 0"39

0"90 0"75 0'54 0'78 0"94 0"02 0"06 0'10

0.36 0.44 0.51 0.57

t Per cent of acetone in petroleum ether b.p. 60--70°C.

between 98 and 100 per cent has been obtained in quantitative determinations, lva Circular chromatography on kieselguhr and alumina paper, allowing simultaneous separation of a considerable number of carotenoids, is characterized by high resolution, by rapidity, ease of operation and high pigment recovery. Co-chromatography on circular paper of the stereoisomers of an unknown carotenoid, with the stereoisomers of a known pigment as obtained by iodine catalyzed isomerization, forms one of the most reliable methods for identification of a carotenoid. The method has been widely used by the present *For example the filter papers Nos. 287, 667 and 996 produced by Carl Schleicher & Schiill, Dassel, Germany.

138

Recent Progress in CarotenoidChemistry authors, and a number of new carotenoids have been discovered through this technique.223, 16a, 237 Recent reviews of the application of paper chromatography to the carotenoid field have been presented by Sestar, 287 by Hager,1~2 and by Jensen. 17° 2. Countercurrent distribution Liquid-liquid partition methods have considerable traditions in carotenoid chemistry. The classical work is that of Willst~itter and Stoll, TM who managed to isolate pure carotenoids from mixtures by this technique. Petracek and Zechmeister259 have determined the partition behaviour of a large number of carotenoids and provided some of the information necessary to set up solvent systems for Craig-distributions of carotenoids. Before this, however, Curl s6 adapted the countercurrent distribution method of Craig to the problem of separating carotenoid pigments. The method, which is based on distribution of the pigments between immiscible organic solvents, will separate carotenoid mixtures into sub-groups showing different solubility characteristics. Thus, carotenes, monohydroxycarotenes, dihydroxycarotenes, monoepoxy-, monoepoxymonohydroxy-, diepoxy-, diepoxydihydroxycarotenoids, and polyhydroxycarotenoids are separated more or less completely by 100 to 240 transfers in a Craig apparatus. Curl56, 66, 62 has developed several different solvent systems and has applied the method with much success to the difficult problem of separating complex carotenoid mixtures from fruits like oranges, lemons, peaches, prunes, persimmons and peppers. 56, 62, 69, 63, 61, 65 The grouping into iso-distributive pigments must in these cases be followed by adsorption chromatography in order to isolate the single components of the mixtures. Generally speaking, the method of countercurrent distribution forms a gentle alternative to column chromatography for an introductory separation of complex mixtures of carotenoids. It requires, however, rather expensive equipment. The method can obviously be developed further by use of the steadystate apparatuses which have recently been made commercially available.4, 96 This will require even larger capital investments but will offer several important advantages over the ordinary Craig apparatus, the continuous operation being the most important feature. B. Characterization of carotenoids A successful isolation and separation procedure should leave the investigator with a number of fractions, each containing only one single carotenoid. When possible, each pigment should be crystallized from suitable solvents until constant properties are obtained.19° Characterization by elementary analysis (C, H, O) and by melting point may then be carried out. As means for the identification ofcarotenoids these characteristics are, however, not very valuable. Better data are obtained from partition coefficients, chromatographic properties, 139

Progress in the Chemistry of Fats and other Lipids light absorption spectroscopy in visible and infrared regions and from nuclear magnetic resonance spectra. It should be pointed out that the extinction value in visible light sharply reflects the purity of a carotenoid, and extinction coefficient determination forms one of the best purity tests available for carotenoids. Since the extinction coefficients of the various chromophores may be predicted rather precisely (see below), this property is most useful in studies of known as well as new pigments.

1. Partition coefficients The original qualitative description of the distribution behaviour of carotenoids when treated with two immiscible solvents like petroleum ether and aqueous methanol has been given a quantitative basis by Petracek and Zechmeister. 259 On partition between petroleum ether and 95 per cent aqueous methanol, the carotenes enter the epiphase and the dihydroxy- or more polar carotenoids are found in the hypophase. Monohydroxy- and similarly polar pigments show intermediate behaviour. The introduction of the N100-values (tube number of the maximum of the pigment after 100 transfers in a Craig machine) by Curl ~6 is a further refinement of the method. The original list of pa~ tition coefficients of Petracek and Zeichmeister has been extended by Krinsky and Goldsmith 2°5 and by Liaaen-Jensen. 227 The latter author has demonstrated how the hypophasic character of an aliphatic carotenoid increases with the length of the conjugated carbon-carbon double bond chain, and was also able to show that neither isolated double bonds nor the character of the hydroxy groups influenced the partition ratio significantly. Partition behaviour in standard conditions therefore forms a valuable tool in carotenoid analysis, especially for characterizing hydroxy- and less polar compounds. With increasing polarity differentiation becomes less distinct. 2. Chromatographic properties The chromatographic behaviour is itself a good means of characterizing a carotenoid and has been made use of extensively.190, 809 The application of paper-chromatographic techniques to the carotenoid field has made it possible to ascribe specific Rf-values to the different pigments, as shown by the present authors 173 and by Jeffrey. 165 Thin-layer chromatography also constitutes a very efficient tool for the characterization of carotenoids, ~97, 298 although reproducible RI values are not easily obtained by this method. Some carotenes are, however, better separated by thin-layer than by paper chromatography, in which cases the former method may be preferable to the latter for the characterization of the pigments. The Rl-values (see Table 2) generally parallel the partition characteristics. The carotenes show the highest and the polyols the lowest rate of migration. Within comparable series of compounds the R~-values decrease with increasing length of the conjugated chain. Alicyclic compounds migrate faster than the 140

Recent Progress in Carotenoid Chemistry

corresponding aliphatic ones. Carotenoids containing secondary hydroxyl groups are more strongly retained than those with tertiary hydroxyls. The keto group leads to a small drop in the Ri-value when comparison is made with the corresponding hydrocarbons. According to the experience of the writers, very few carotenoids indeed are inseparable by circular paper chromatography, and we have never encountered any stereoisomeric set that completely overlapped with a non-identical set. As pointed out above and discussed elsewhere, s27, 17o co-chromatography on paper of stereoisomeric sets (after iodine catalyzed isomerization) forms one of the most discriminating identity tests available to the chemist working with carotenoids.

3. Light absorption properties in the visible and ultraviolet regions The connection between colour and constitution, so easily observed with carotenoids, has attracted the interest of many scientists. Karrer and Jucker19° have reviewed the literature available up to 1950. Since then a considerable body of new evidence has deepened our understanding of the chromophoric systems of carotenoids. 2O

A

I

'":J'¢i l i

,,

,J 200

300

~ 300 m,u.

\\ 500

FIG. 1. Spectra of . . . . . phytofluene (H), - - ' - - neurosporene (IV), lycopene (V) and . . . . . spiriUoxanthin (XXVIII), demonstrating the influence of chromophore prolongation on spectral properties.

The spectrum in visible light of a carotenoid is first of all determined by the number of conjugated double bonds in the chromophore (see Fig. 1). Karrer and Jucker19° ascribed a constant increment in the A max of the pigments to each aliphatie carbon-carbon double bond added in conjugation to an aliphatic 141 L

Progress in the Chemistry of Fats and other Lipids

Table 3. Spectral effect on various prolongation o f the carotenoid chromophore i

Type of ehromophore

Compound

Number of ! AC R conj, C = C in mlz

Aliphatic Aliphatic Aliphatic Aliphatic Aliphatic Aliphatic A liphatic

Phytoene Phytofluene ~-Carotene Neurosporene Lycopene Dehydrolycopene Bis-dehydrolycopene

3 5 7 9 11 13 15

351 26 l 201 181 13l 81 7t

Alicyclic, normal Alicyclic, normal Alicyclic, normal

a-Carotene fl-Carotene ~,-Carotene

11 II 11

7z 6e 52

Alicyclic, retro

Retrodehydro-fl-carotene

12

92

Aliphatic, Aliphatic, Aliphatic, Aliph~ttic, Aliphatic,

mono-carbonyl mono-carbonyl mon>carbonyl mon>carbonyl mono-carbonyl

Apo-4-i3-carotenal a-Citraurin Apo-2-a-carotenal fl-Citraurin Apo-2-1ycopenal

7 8 8 9 10

2Tz 28 a 283 27:; 29 a

Aliphatic Aliphatic Aliphatic Aliphatic

di-carbonyl di-carbonyl di-carbonyl di-carbonyl

Crocetin dialdehyde Capsylal Capsanthylal /3-Carotenone

7 7 8 9

24 54 74 14

11 11

55 96

11 12

77 56

Alicyclic, mon )-carbonyl, normal Alicyclic, mon>carbonyl, retro Alicyclic, di-carbsnyl, normal Alicyclic, di-carbonyl, retro

Echinenone 3-Keto-retrodehydro-flcarotene i Canthaxant-hin i Rhodoxanthin

1 Increment of the last carbon-carbon double bond in the aliphatic chromophore. 2 Increment of the carbon-carbon double bond in the ring. 3 Increment of the first carbon-oxygen double bond in an aliphatic chromophore. 4 Increment of the second carbonyl group in aliphatic systems. Average value of the first carbonyl is 25 m/~. 5 Increment of the first carbonyl in the ring of a "normal" carotenoid. 6 Increment of the first carbonyl in the ring of a retro carotenoid. 7 Increment of the second carbonyl in a normal carotenoid. s Increment of the second carbonyl in a retro carotenoid. c h r o m o p h o r e , irrespective o f the l e n g t h o f the latter. D a l e 70 s h o w e d in a d i a g r a m h o w this i n c r e m e n t decreases f r o m m o r e t h a n 25 to less t h a n 10 mt~* as the n u m b e r o f c o n j u g a t e d d o u b l e b o n d s increases f r o m 5 to 15 (see T a b l e 3). T h i s applies to a l i p h a t i c systems, w h i c h exhibit p r o n o u n c e d fine s t r u c t u r e spectra. T h i s fine s t r u c t u r e increases w i t h i n c r e a s i n g n u m b e r o f d o u b l e b o n d s u p to 9 c o n j u g a t e d b o n d s . F u r t h e r e x t e n s i o n o f the c h r o m o p h o r e leads to slight loss in * In the present treatise all spectroscopic data refer to solutions of the pigments in petroleum ether. 142

Recent Progress in Carotenoid Chemistry fine structure. When the chromophore is extended into the rings of alicyclic pigments, the additional double bond counts for approximately ~ of the corresponding aliphatic double bond in the "normal" series. (See Fig. 2. In "normal" carotenoids the cyclohexenyl rings are linked to the central chain by single bonds, while double bonds occupy these positions in the " r e t r o " pigments.)

J l 151

. . . . . . . . . .

0

/

i,i

I

l

/ /

L

i

300

400

500

m/z

Fic. 2. Spectra of . . . . . lycopene (V), - - - - "e-carotene(IX) and . . . . . /3-carotene (XIII), showing the spectral effect of cyclization. In the retro carotenoids the double bond in the ring is somewhat more efficient in lengthening the effective chromophore. By extending the chromophore into the rings, the fine structures typical of the aliphatic pigments are reduced markedly in the "normal" series. The retro pigments exhibit, however, good fine structure spectra. Theoretical interpretations of the fine structures in the spectra of polyenes have been given by Dale, 7°, 71 Platt265 and by Kuhn. 206 The introduction of carbon-oxygen double bonds in conjugation with a polyene chromophore has a marked effect upon the spectral properties of the pigment. The carbonyl group produces a bathochromic displacement of the absorption maximum of about 25 mt~ when this function is introduced into one 143

Progress in the Chemistry of Fats and other Lipids end of an aliphatic system or at the aliphatic end of an alicyclic chromophore (see Fig. 3). The length of the polyene chain has little influence on the increment of the added carbonyl group. A carboxyl ester group added in conjugation to such polyenes shifts the absorption maxima by some l0 to 15 m/~ in the case of lower homologs of torularhodin methyl ester, 143 whereas the shift in azafrin methyl ester is larger than 20 mt~.2°9 sooo .

!

I

I. L

I J ,/

/! /

/

iooo

/

\

/

t

1

/

/!/

500

-

/A/

560

F I G . 3.

7-7

/i

400

l'~X/

/

\

. . . . . . .

t \

5

\

450 mff

t t

500

\

550

Spectra o f - - - - OH-spheroidene (XXI) and . . . . . OH-spheroidenone (XXXVII), showing the spectral effect of a conjugated keto group in an aliphatic chromophore.

When a carbon-oxygen double bond is introduced into a ring of a cyclic carotenoid and in conjugation with the polyene chain, the bathochromic shift will be smaller than in aliphatic systems, TM some 5 and 9 mt~ respectively in the two series /3-carotene: echinenone, z32 and retrodehydro-fi-carotene: 3-ketoretrodehydro-[3-carotene 8s (see Fig. 4). The effect of two carbonyl groups in conjugated oJ,~o'-systems has caused considerable discussion. 351 In the case of aliphatic carotenoids, the second carbon-oxygen double bond does not shift the light absorption maximum much towards longer wavelengths, when comparison is made with the corresponding 144

Recent Progress in CarotenoidChemistry mono-carbonyl polyenes (see Table 3). Bathochromic shifts in the light absorption spectrum of about 1-7 mtz are frequently encountered. In the series of dicarboxylic carotenoids, two carboxyl ester groups produce a bathochromic shift of approximately 20 mr* together, e.g. in compounds like crocetin dimethylester and methyl bixin. The difference in effect of carboxylic ester contra carbonyl groups is clearly demonstrated in the series crocetin dimethylester (diester Z~max 3000

._'j 1000

400

500 rnp.

FIG. 4.

Spectra of - -

Jsozeaxanthin

(4,4'-dihydroxy-fl-carotene)

and

.....

canthaxanthin (4,4'-diketo-~-carotene), demonstrating the spectral effect of conjugatedketo groups in cyclicsystems. 420 mr0: azafrinone (keto-methyl ester Area,, 427 mtz): /3-carotenone aldehyde (keto-aldehyde ¢~max431 m/z) or crocetin dialdehyde (dialdehyde)tmax 428 rntz). In bicylic dicarbonyl carotenoids with both carbon-oxygen double bonds in the rings and in conjugation with the polyene chain, both carbonyl groups seem similarly active for the extension of the chromophore. Going from/3-carotene, through echinenone (4-keto-/3-carotene) to canthaxanthin (4,4'-diketo-/% carotene), the shifts are 5 and 7 m~ respectively. In the series retrodehydro-flcarotene, 3-keto-retrodehydro-~-carotene to rhodoxanthin (3,3'-diketo-retrodehydro-/3-carotene), the corresponding shifts are 9 and 5 mt~ respectively. Concerning the effect of carbon-oxygen double bonds in the conjugated chromophores of carotenoids, the rule will thus be that one carbonyl group (a,/3-conjugated) produces a bathochromic shift of approximately 25 mtz (10-15 rn~ for carboxylic acids), whereas a second carbonyl function in ~o-position leads to a small shift only. An exception to this rule is found within the cyclic pigments, where introduction of the first carbonyl group also results in only a small bathochromic shift. The molar extinction coefficients of the absorption bands in visible light vary from 77.3 x 103 for phytoene to 185-5 × 103 for lycopene, which represent the two extremes. Upon lengthening of the chromophore beyond that of lycopene a drop in the extinction value is observed. When the conjugated systems enter into the rings of cyclic carotenoids, the intensity of the absorption bands in 145

Recent Progress in Carotenoid Chemistry visible light is likewise reduced markedly, especially in the normal series, as is easily seen from a comparison of the molar extinction coefficient of the aliphatic lycopene (185.5 × 10a) with those of the monocyclic 7-carotene (166.2 × 103) and the bicyclic fi-carotene (137.2 × 103). In retro pigments this drop is less pronounced. Retrodehydro-fl-carotene has a molar extinction coefficient of 171.8 × 10a. Introduction of conjugated carbonyl groups has considerable effect on the molar extinction coefficient of both cyclic and aliphatic systems. Thus the molar extinction value drops some 20 per cent when fl-carotene is oxidized to echinenone (4-keto-fl-carotene) and when 7-carotene is compared with apo-4'-fi-carotenal. Also in retro systems, conjugation with carbonyl groups leads to a similar reduction in the intensity of the absorption bands in visible light. The 3-ketoretrodehydro-fi-carotene of Entschel and Karrer s5 exhibits an extinction coefficient of 141.5 x 10a compared to 171.8 × 10~ for retrodehydro-fi-carotene itself. Hydroxy-containing carotenoids compare well with their parent carotenes in some cases. In the series fi-carotene :cryptoxanthin :zeaxanthin molar extinction coefficients are almost identical. However, when 1,2,1',2'-tetrahydro-l,l'dihydroxy-lycopene, rhodopin and lycopene are compared, a drop in molar extinction value (intensity of the absorption band) is observed with the introduction of new hydroxyl groups. Within stereoisomeric sets the all-trans compounds invariably show the highest extinction coefficient of the main bands in the visible part of the spectrum. Upon isomerization of one or more trans-olefinic bonds in a carotenoid the absorption maxima in visible light often undergo a hypsochromic shift of variable magnitude, followed by a drop in the intensity of the peak. A full discussion of these phenomena is given in Zechmeister's book on cis-trans pigments, aS'~ A remarkable hypsochromic shift of some 40 m/x and a striking drop in intensity is observed in the case of synthetic pigments with internal steric hindered cis bonds. 2s7, 87,142 In the near ultraviolet region of the spectrum the carotenoids exhibit minor absorption bands which may give valuable information. This includes the socalled "cis-peak" region. Dale 71 has discussed the relationship of the minor bands and has stated that these bands are "overtones" of the whole chromophore and not due to excitation of parts of the chromophore. A theoretical treatment of the light absorption in polyene systems has also been presented by Kuhn. 2°6, 207 Zechmeister 352 has published a very extensive discussion of the cis-peaks, and points out that all trans-caroteno!ds lack this absorption, whereas most cisisomers obtained by catalytic isomerization do exhibit cis-peaks. All cis-peaks of a given stereoisomeric set are located at the same wavelength and always at a nearly constant distance from the main maximum of the all-trans isomer. Naturally occurring polycis-compounds, in common with the cis-isomers of retro pigments do not show cis-peaks. 146

Progress in the Chemistryof Fats and other Lipids Carotenoids with aromatic end groups, like renieratene (XVII), isorenieratene (XVI), renierapurpurin (XVIII) and chlorobactene (XII), exhibit gross spectral properties confusingly similar to those of the alicyclic pigments. The shifts in light absorption maxima, caused by addition of aromatic groups to the polyene chain, varies with the substitution pattern of the aromatic nucleus. Thus the 1,2,3-trimethyl substituted benzene ring equals one aliphatic double bond and the 1,2,5-trimethyl compound corresponds to ½ of this, as does the so-called fl-endgroup (fl-ionone ring). The latter fact unfortunately does not permit a differentiation of aryl-carotenoids on the basis of the visible light absorption spectrum. In passing, attention is drawn to the small but significant hypsochromic shift and the slight loss of fine structure in the spectrum of fully deuterated carotenoids observed by Strain, Thomas, Crespi and Katz.at3 4. Absorption spectra in infrared light Infrared spectroscopy has been frequently applied to the field of carotenoid pigments. Because of the relatively recent practical development of this method these applications have not been reviewed before. In Table 4, therefore, a fairly complete list is given of the naturally occurring carotenoids for which IR-data is to be found in current literature. Several techniques have been used for obtaining the spectra. The most suitable solvent systems seem to be carbon tetrachloride, chloroform (2-12/~), bromoform, carbon disulphide, and cyclohexane (12-15 ~). Nujol mulls have also been employed.314 However, the ordinary potassium bromide pellet technique gives very good spectra and may be carried out in sub-micro scale. As little as 0.2 mg of the pigment is suflicient. 22s Strain eta/. ala, 314 obtained good infrared spectra with melts between potassium bromide plates. This technique must, however, be carried out with the utmost care, since most carotenoids tend to undergo isomerization and/or destruction upon heating to the melting point. Differences in the O--H stretching region (free and bounded OH), and in the region influenced by crystal aggregation (,-~950/z) may be expected when spectra of solutions are compared with those of the same compound in the solid state. 314 The infrared spectra of the carotenes and the hydroxycarotenoids are surprisingly simple (see Fig. 5). The limitation of the method for characterizing such compounds is clearly indicated by the fact that the aromatisity in the Reniera carotenes may hardly be detected in the spectra, 237 and compounds like /3-zeacarotene and torulene show practically identical infrared spectra, although the former has 9 and the latter 13 conjugated double bonds in the chromophore. This having been said, it can be stated that infrared spectroscopy is, indeed, a valuable tool in structural studies of carotenoids. More general discussions of the spectral features of common carotenoids have been given by Lunde and Zechmeister245 and by Strain et al. 31a, 314 A brief discussion of the absorption bands associated with structural groups characteristic of, or frequently occurring in carotenoids will be given here.

147

Progress in the Chemistry of Fats and other Lipids

Table 4. Sources of iqf?ared data of naturally occurring carotenoids Pigment

Reference

Anhydro-rhodovibrin Astacirt Astaxanthirt a-Bacterioruberin Castthaxanthin Capsanthin Capsorubin ~-Carotene /3-Carotene ~,-Carotene ~-Carotene Chlorobactene OH-Chlorobactene Chloroxanthin Cryptocapsin Cryptoxanthin Deuterio-a-carotene Deuterio-/3-carotene Deuterio-lutein 2,2'-Diketospirilloxant hin Dimethylcrocetin Echinenone Eschscholtzxanthin Fucoxanthin Isorenieratene (= Leprotene) Lutein Lycopene Lycoxanthin Methylbixin Monodemethylated spirilloxanthin Mutatochrome Neurosporene Okenone Physaliene Phytoene Phytofluene Renierapurpurin Renieratene Rhodopin Rhodovibrin Rhodoxanthin Spirilloxanthin Spheroidene OH-Spheroidene Spheroidenone OH-Spheroidenone Torulene Violaxanthin /3-Zeacarotene Zeaxanthin

148

227 219, 75, 22 219 227,225 332 331, 13, 12 331, 13, 54 245, 313,314 245, 140,315, 149, 313, 314 245, 279 74 237,24 24 227, 222 45 147, 314, 22 313 313 313 228 245,145 332 314 219, 323,168 53,344 313,314,22,227 245, 140, 314, 144 227, 223 245, 145 230 2 [, 22 74 286 140 74 74 53,345 53, 345 227, 223, 281, 24 227, 224, 230 219, 22 227, 221,228, 230, 14 221,230 230 113, 254, 228, 229 229 28O 222 28O 245, 148, 222, 22

Recent Progress in Carotenoid Chemistry

The carbon-carbon double bond. Practically all infrared spectra of carotenoids contain a strong band or group of bands near 965 cm -1. According to Lunde and Zechmeister, 245 this absorption peak is associated with the out-ofplane vibration of C - - H in the trans configuration of the - - C H = C H - - grouping. The corresponding C--H-stretching mode of vibration is found at approximately 3,030 cm-1. 3~3 The all-trans-forms of carotenes belonging to the normal series are supposed to show one peak only in the 960 cm -1 region. Some central cis-carotenes, z45, 74,149 however, exhibit doublets in this area. Splitting of the "trans peak" otherwise occurs in the spectra of the retro carotenoids. In the spectrum of eschscholtzxanthin, 314 two bands are reported (at 955 and 977 cm-1); and both dehydroretro-carotene and 4,7-dihydro-dehydro-retrocarotene show peaks near 960 cm -1 and 980 cm-~. ~54 This splitting is obviously a characteristic feature of the aliphatic polyene chain andis not affected by the conjugation of this with cyclic end groups of the retro form. 154

'~'

i ....

i

V

i

i

i

i

i___

3~

!i

FIG. 5. Infrared spectrum of a typical bicyclic carotenoid with secondary hydroxy group (isozeaxanthin = 4,4'-dihydroxy-fl-carotene). 15°

A third group of carotenoids that exhibit complex absorption in the I000 to 900 cm -1 region is composed of the pigments containing conjugated carbonoxygen double bonds. Thus, dimethylcrocetin and methylbixin245, 145 show several peaks in this region. Capsanthin and capsorubin have triplets near 1,007, 980 and 970 cm-1. 331 Spheroidenone and OH-spheroidenone exhibit doublets228, 229 which disappear on reduction with lithium aluminium hydride, which proves that the complexity of the infrared absorption in that region is caused by the presence of the ketopolyene conjugation. Tri-substituted double bonds are always present in carotenoids and cause weak absorption around 830 cm -1 ( C - - H out-of-plane deformation). The presence of cis-carbon-carbon double bonds in carotenoids gives rise to a strong absorption band near 750 cm -1 (out-of-plane vibration of C - - H in the c i s - C H = C H - - grouping 245) in the case of "unmethylated double bonds", while a band near 1380 cm -1 has been assigned to the deformation vibration of the methyl group on a cis-double bond in aliphatic systems. 245 A similar 149

Progress in the Chemistry of Fats and other Lipids absorption peak in all-trans-a-carotene was explained as originating in an analogous vibration of the methyl group on the double bond in the ring. 245 Hydroxy-earotenoids. The O - - H stretching vibrations of xanthophylls give rise to infrared absorption bands at 3650-3320 cm 1. In carbon tetrachloride, the peak corresponding to free hydroxyl groups (3,644 cm -1) is sharp, while that caused by associated hydroxyl functions is small (3,400 cm-1). 314 In potassium bromide pellets, the peak near 3400 cm -1 becomes prominent. 224, 225,314 Xanthophylls containing secondary hydroxyl groups exhibit a characteristic absorption peak of medium intensity in the 1025 to 1045 cm 1 region for cyclic222, 227 (see Fig. 5), and near 1010 cm -1 for aliphatic 223 compounds, while the hydroxy carotenoids with tertiary hydroxyl groups give rise to weak absorption bands at 1150-1130 cm 1 and near 905 cm -1 227,222,224,230 (see Fig. 6). It is therefore possible to differentiate between secondary and tertiary hydroxyl groups in carotenoids by means of infrared spectroscopy. Bodea et al. 22 have shown that a number of cyclic carotenoids bearing a hydroxyl group in the 3-position (in fi-position to an olefinic bond) exhibit C - - O H vibration bands at 1045 cm 1, while the corresponding peak was found at 1030 cm -1 in the case of several xanthophylls with the hydroxyl group in the 4-position (allylic hydroxyls). The carbon-oxygen double bond. Carbonyl groups are frequently encountered in the naturally occurring carotenoids, and almost invariably in conjugated systems. The esters of xanthophylls are exceptions, as the carbon-oxygen double bond of the esterifying acid is never conjugated with the polyene chain in any pigment known today. The stretching frequency of the unconjugated ester carbonyl function is usually found near 1735 cm-1.140, 219,323,168 Upon conjugation with the polyene chain the carbonyl absorption peak usually moves to the 1680-1640 cm t region (see Fig. 7). Typical examples are canthaxanthin (1667 cm-1 22), rhodoxanthin (1669 cm -1 22,219), spheroidenone (1680 cm -1 22s, 229), capsorubin (1664 cm 1 331, 13, 54), capsanthin (1661 cm -a 311, 13, 12), cryptocapsin (1664 cm -1 45) and fucoxanthin (1635 cm -1 219,323, 16s). The diosphenol grouping in astacin absorbs at 1626 cm -1 and 1560 cm l 219 and thus falls outside the normal range of conjugated carbonyl compounds. An overtone of the carbonyl stretching band is found at 3320 cm -1 in, for instance, spheroidenone, and should not be mistaken for a hydroxyl absorption. Like other carbonyl-compounds, carbonyl-containing carotenoids also exhibit medium strength absorption in the 1250 cm -1 region. Conjugated polyene ketones (and carboxylic acids) show much stronger absorption in the C - - C stretching region than do the corresponding carotenes (see Figs. 6 and 7). Several strong bands between 1590 and 1550 cm -~ occur in pigments like capsorubin, capsanthin, spheroidenone and fucoxanthin. These bands disappear upon reduction of the keto group. 229, 169 Another feature frequently encountered in the group of conjugated keto carotenoids is the splitting of the absorption peak caused by the C - - H out-ofplane deformation vibration of the trans-CH CH grouping, as mentioned 150

Recent Progress in Carotenoid Chemistry

O

~× o_

Q

,xZ ,~:z

7

~6 t~

8o -

i

o uo!J,dJ0sqv

151

8

Progress in the Chemistry of Fats and other Lipids

t

o, 0 OF

I

.....

!

e~

o! I

f

-zk

0

8~ ~= 'S .~,>

T

E!

o©

E~

I, 1

oi

.=2:

S c~

o

o

o

o

uo!~dJ0sqv

152

Recent Progress in Carotenoid Chemistry above for the compounds dimethylcrocetin, methylbixin, capsanthin, capsorubin, spheroidenone and OH-spheroidenone. The methoxy group. In the series of bacterial carotenoids which carry methoxy groups in the 1 and 1" positions, 232 the C - - O stretching vibration of the ether group gives rise to a band of medium intensity at 1080-1070 cm -1 227,229,230 (see Fig. 7). The corresponding absorption peak of the synthetic 4,4'-dimethoxyfi-carotene occur at 1097 and 1082 cm -1 (see ref. 39 in 227). Other groups. Carotenoids containing the hydrofurano grouping are said to show characteristic absorption near 1070 cm-1. 21, 22 5,6-Epoxy-carotenoids like violaxanthin have no reasonably strong absorption band which can be ascribed to the epoxy groups. The very unusual peak at 1935 cm -1 in the infrared spectrum of fucoxanthin 219, 323,168 has been assigned to an allene grouping. In hydrogenated derivatives of carotenoids the central chain of 4 methylene groups may give rise to a broad band near 750 cm -1. Aromatic carotenoids exhibit a weak, but characteristic absorption band at 800 cm -1 for the C - - H out-of-plane deformation of the tetra-substituted benzene ring. The C = C skeletal in-plane vibration for the benzene ring is not sufficiently strong to give characteristic absorption for aryl-carotenoids in this region. 5. Nuclear magnetic resonance spectra The development of nuclear magnetic resonance spectroscopy into a routine technique requiring as little as 1 to 10 mg of material, has added a new and most powerful tool to the list of useful micro techniques available to the organic chemist. The first systematic application of n.m.r, spectroscopy to the field of carotenoids was reported by Barber, Davis, Jackman and Weedon 11 who carried out an extensive study of the signals given by the different methyl groups of 64 carotenoids and related compounds. Interested readers are referred to this very valuable and fundamental study. A feature common to the spectra of all the carotenoids was found to be a band in the region 7.95-8.15 (positions of the bands are quoted as ~--values) assigned to the "in-chain" methyls of the conjugated polyene chain. Many carotenoids show only one such "in-chain" methyl band, which is located at 8.03 in /3-carotene. Modifications of the chromophore may cause splitting, broadening, or deviations from the normal position as exemplified by azafrin and apo-4-carotenal, which have two "in-chain" methyl bands. Upon conjugation with an aromatic system, the "in-chain" methyl group 7 to the phenyl group gives a band at 7.92-7.96. 53 The bands associated with methyl groups attached to the terminal carbon atoms of the polyene chromophore ("end-of-chain" methyls) are found at higher fields; in lycopene at 8.18, in/3-carotene at 8.30, and in mutatochrome at 8-268.28. "End-of-chain" methyl groups which are in a-position to a carbonyl function give rise to bands in the region 7.98-8.13 (in aliphatic esters and aldehydes). 153

Progress in the Chemistry of Fats and other Lipids The isopropylidene end group is characterized by a doublet at 8.38 and 8.31 in aliphatic carotenoids like lycopene. In cyclic pigments of the fi-carotene type the gem-dimethyl grouping gives rise to one band only at 8.97, while in the pigments with a-ionone rings, two bands, at 9.14 and 9-08, may be assigned to the gem-dimethyl groups. In the case of epoxides, the gem-dimethyl groups are influenced to different extents by the proximity of the polyene chain and give rise to separate bands around 9.10 and8.90. Also the methyl group in the 5-position of these compounds is influenced by the oxide formation, and the corresponding band is found near 8.90. The difuranoid oxide aurochrome exhibited a striking band at 4.84, which was assigned to the four protons of the two heterocyclic rings. Methoxy groups, as found in spirilloxanthin, give rise to a band at 6.78.14 Extensive use of n.m.r, spectroscopy has been of great value in the structural elucidations of carotenoid pigments like capsanthin, capsorubin, 12, 13,54 spirilloxanthin, 14 cryptocapsin, 45 phytoene, phytofluene, {:-carotene and neurosporene, 74 3,4-dehydro-rhodopin, 163 chlorobactene and OH-chlorobactene 24, 237 and OH-spheroidene, OH-spheroidenone and 2,2'-diketo-spirilloxanthin. 16~ The n.m.r, spectrum of the methyl ester of natural bixin 15 has given the conclusive evidence for placing the cis-double bond of this pigment in the 6-position, and has thus brought the discussions of this disputed problem to an end. In the n.m.r, spectra the aromatic character of the Reniera carotenoids and of the chlorobactene and OH-chlorobactene is easily detected, since the methyl groups on the aromatic ring give characteristic signals in the 7.70-7.85 region. Also, the aromatic protons are characteristic and give rise to bands near 3.04. 237 To summarize the more important n.m.r, data mentioned above, the structures of five different carotenoid pigments are given in Fig. 8, together with the -r-values connected with the various structural units for which assignments have been given in the literature. As may be seen from Fig. 8, olefinic protons fito a carbonyl group, or on aromatic rings, as well as methylene groups in different surroundings, give rise to characteristic signals. It is obvious that small structural modifications may have strong effects on band position and splitting, and the interpretation of the n.m.r, spectrum of an unknown carotenoid has to be carried out with great caution and in conjunction with other physical and chemical evidence, as pointed out by Barber, Davis, .lackman and Weedon. 11

6. Stereochemistry Cis-trans isomerization. The cis-trans isomerization of carotenoids, vitamins A and arylpolyenes has been expertly treated in a recent book by Zechmeister 352 to which the reader is referred. Only a brief discussion, therefore, will be delivered here. In theory all the double bonds in the aliphatic chain of the carotenoids can occur in the cis or the trans configuration. Of the many isomers possible only some few occur in considerable quantities within each set of stereoisomers, and, as a rule, the all-trans member is by far the most frequently occurring form 154

Recent Progress in Carotenoid Chemistry in natural products. It is also normally the most stable form. It should be emphasized, however, that all carotenoids known so far, do isomerize to variable extents, when illuminated in solution, especially in the presence of a catalyst like iodine, and that a considerable number of cis-carotenoids have been found in nature. The tendency to isomerize increases when one goes from the bicyclic to the monocyclic and the aliphatic pigments. 8.g7

8.97

~

8.03

I~03

~

8.38

/P~

8.03

8.03

8.18

831

"8.30

V

~ CaPo~ene (279)

6.79 iH c o \

8.G.s /

8.01 1

0,01 1

8.01 /

7.e5 ~

7.85

8.79

1 3e,65

OH 0

8.01

8.76

8.76

8.03

6.03

839

8.7g

(164)

OH- SphePoldenonone

HO

B.17

8,03

7.86

8.39

7.70 8.03

8.03

8.19

B.32

3 . 4 " DehydeoehodOp~n (163) 7.43

8,03

f ~/'x. ~ , ~¢t" ~ ~

a.o3

2 x 7 , 7 7 , 7.75

....

~.,9

8.32

Chloeobactene (237)

8.02

~l. 6 3 ,

8.o3

OH . , 8.80~ 9.15

o.2.8/N.

8.02

"~ "~I

CPypt ocapsirl [45)

FIG. 8. Nuclear magnetic resonance signals for some carotenoids. The aU-trans carotenoids invariably absorb visible light at longer wavelength than the other members of the stereoisomeric set, and also show the highest extinction values. Thus, isomerization of an all-trans pigment results in a hypsochromic shift in the absorption spectrum and a loss of optical density, while 155

Progress in the Chemistry of Fats and other Lipids

cis-isomers will show bathochromic displacement of Amax and increased extinction value as a result of isomerization. Spectral characteristics of cis-carotenoids in the ultraviolet and infrared regions have been discussed earlier in subsections 3 and 4. Many eis-carotenoids, even those with very much hindered cis bonds, have become available through synthesis during the later years, and this has greatly facilitated the studies within the field. A number of the members of a stereoisomeric set may be separated by column chromatography. Paper chromatography of equilibrium mixtures (obtained by iodine catalyzed isomerization) forms a good method for the identification of the parent carotenoids (see page 138). Configuration studies. This is a much neglected field in carotenoid ehemstry. The configuration of the many asymmetric and optically active compounds of this class has been established in only a very few cases. The steric arrangement around the frequently occurring 3-hydroxy grouping in xanthophylls has recently been assumed to be of the a-type. 54 This assumption was based on the biogenetic relationship between zeaxanthin and the paprika ketones, capsanthin, capsorubin and cryptocapsin. The full stereochemistry of capsorubin and cryptocapsin has been established by means of n.m.r, and infrared data and by degradation and synthesis. TM 93, 45 The absolute configuration of the cyclopentanone ring in capsanthin is very probably the same as that in cryptocapsin.45, 93 Beside this very little is known about the configuration of carotenoids (other than cis-trans isomerization of the double bonds). Karrer and Jucked 90 have indicated that flavoxanthin and chrysanthemaxanthin are epimers around the hydroxy group in the 3-position of ring A. OH

|

Crysanthemaxanthin,

flavoxanthin

In the synthesis of cryptoxanthin, zeaxanthin and physalien,147, 148 racemic forms have been reported, and the synthesis of iso-zeaxanthin153 gave one mesoform and one racemate. These two groups could be separated from each other by chromatography. No further separation or study of the stereochemistry was undertaken. Akhtar and Weedon (see ref. 11) assumed that the vic-dihydroxy system in methyl azafrin will favour a conformation with the two hydroxyl groups in equatorial position. The data given above clearly demonstrate the validity of the statement made by Karrer and Jucker 189 in 1948 in connection with the steric isomerism of flavoxanthin and chrysanthemaxanthin: "The question of this isomerism requires further investigation." 156

Recent Progress in Carotenoid Chemistry

C. Group reactions 1. Carbon-carbon double bonds By definition, a considerable number of carbon-carbon double bonds are present in all carotenoids. This number may be determined by quantitative hydrogenation, and the number of conjugated double bonds be deduced from the light absorption properties of the pigment in the visible region of the spectrum (see Section II, B3). Certain types of cis-double bonds may be seen in the infrared spectrum. The double bond in iso-propylidene end groups is determined quantitatively by the K u h n - R o t h procedure. 175, 227,274 It has been shown that carotenoids carrying hydroxy or methoxy groups in the 1,1'-position give rise to a false iso-propylidene value. 227

2. Hydroxy groups The hydroxy groups in xanthophylls reveal their presence already during the isolation and purification procedures, as the pigments become more hypophasic with increasing number of hydroxy groups, and show low Ri-values when the number of hydroxy functions increase to two or more (see preceding sections on chromatographic properties and partition coefficients). Hydroxy groups are easily detectable by infrared spectroscopy by the absorption in the 3650-3320 cm -1 and l l 5 0 - 1 0 1 0 c m -1 regions. As wasmentioned earlier, different spectra in the latter region are obtained for tertiary and secondary alcohols. The tertiary character of a hydroxy group may be revealed through the resistance towards esterification typical of such functions. Whether or not an ester has been formed may be established by means of partition coefficients and paper chromatography since esters behave more or less as their parent carotenes. With sub-micro amounts of xanthophylls, when recording of infrared spectra is not possible, secondary hydroxyl groups may be disclosed by esterification (acetylation at room temperature with acetic anhydride-pyridine is preferred). The course of the reaction may be followed by means of paper chromatography.227,168,169 The presence of allylic (to the polyene chain) hydroxy and methoxy groups can be demonstrated by treatment with acidified chloroform1~2,330, 107,258, 84 which leads to the elimination of water (or methanol) and to the introduction of an additional double bond in conjugation with the polyene chain. The loss of hydroxy groups and extension of the chromophore may easily be detected with sub-micro amounts of pigments (paper chromatography, partition coefficients, absorption spectrum in visible light), and the reaction has recently been used with success for several naturally occurring carotenoids. 229, 128,238 A more detailed discussion of the method has been given by Liaaen-Jensen. 227 Carotenoids containing tertiary hydroxy groups are dehydrated upon treatment with phospho-oxychloride. This leads to the introduction of a new carboncarbon double bond. This reaction has been used in synthetic work 317 as well as for structural elucidation of naturally occurring carotenoids. 237, 2~, 164 157 M

Progress in the Chemistry of Fats and other Lipids Methylation of carotenoid alcohols is discussed in Section IV, 3. Oxidation of the hydroxy groups has also been used repeatedly to demonstrate the presence of secondary alcohol functions in carotenoids. Oppenauer oxidation transforms allylic as well as non-allylic hydroxy groups into the corresponding keto functions, 35s, 12, 45 while p-chloranil has been used by Warren and Weedon z31 to oxidize selectively the allylic alcohol group of reduction products of capsanthin and capsorubin. In model studies of related substances Warren and Weedon had found that the otherwise useful manganese dioxide did not work. Oxidation of zeaxanthin with this reagent is followed by simultaneous dehydrogenation, giving rhodoxanthin (3,3'-diketo-retro-fi-carotene). 85 Vic-diols, like perhydro-azafrin2°9 and 5,6-dihydroxy-/3-carotene,20s are attacked by lead tetra-acetate and by periodate to give the corresponding diketones.

3. Carbonyl groups The presence of carbonyl groups in carotenoids is easily demonstrated by infrared spectroscopy. When the carbonyl group is conjugated with the polyene chromophore, the light absorption properties in the visible region are markedly influenced. Considerable shift of absorption maxima, loss of fine structure and drop in extinction value are observed. The loss of fine structure is particularly pronounced when methanol is used as solvent (see Fig. 9). The influence of the keto group on the Rs-value of carotenoids is much smaller than that of the hydroxy function.

!

SJ

i

............

•

E

o

400

500

600

mp.

FIG. 9. Visible light absorption spectra of spheroidenone (XXXVI) in - - - - - - petro-

leum ether and . . . . . methanol, demonstrating the solvent effect on spectra of conjugated keto carotenoids. 158

Recent Progress in Carotenoid Chemistry

Carbonyl-containing carotenoids may be conveniently reduced to the corresponding alcohols by treatment with lithium aluminium hydride or sodium or potassium borohydride,z29, ~al The reduction is followed by characteristic hypsochromic shifts in the position of the absorption maxima (see Section II, B3), decrease in R~-value and increase in hypophasic character. All these changes may be followed by sub-micro methods like visible light spectroscopy and paper chromatography. Conjugated diketones of the ~o,,o'-type may be reduced to the corresponding dihydro-derivatives by zinc in acetic acid-pyridine, according to Kuhn and Brockmann.~11, lo4 For the specific detection of trace amounts of aldehydic carotenoids in natural products, Winterstein, Studer and RiJeggas8 have introduced a rhodamine reagent in combination with thin-layer chromatography.

4. Epoxy groups and furanoid rings Epoxy groups have only been found in the 5,6-position of cyclic carotenoids. Upon treatment with dilute organic acid they undergo isomerization with ring expansion to hydrofuranoid systems. 308, 6s This isomerization is followed by a large hypsochromic shift in the position of the absorption maxima in visible light (corresponding to the loss of 1½ spectroscopically efficient conjugated double bonds). The furanoid oxides are not affected by treatment with dilute organic acids under controlled conditions.68 Most epoxy-carotenoids and hydrofuranoid pigments give a blue colour reaction with concentrated hydrochloric acid, and this treatment forms the basis of the colour test190 for epoxides and furanoid carotenoids. Modifications in the original procedure have been introduced by Curl and Bailey6s and by Yamamoto, Chichester and Nakayama.346 Attention is drawn to the fact that, according to Tsukida and Zechmeister,z25 the epoxy group may isomerize to a hydrofuranoid ring during illumination of pigment solutions and upon contact (in the dark) with alkaline alumina. Jaeger and Karred 61 have developed a method for the removal of the epoxy function in carotenoids. Upon treatment of diepoxy-fl-carotene with propyl magnesium bromide and ferric chloride,/3-carotene was obtained in 65 per cent yield. Violaxanthin (5,6-5',6'-diepoxy-zeaxanthin) gave 30 per centzeaxanthin and 25 per cent antheraxanthin (5,6-epoxy-zeaxanthin).

III. STRUCTURAL ELUCIDATION OF NEW, NATURALLY OCCURRING CAROTENOIDS

At present approximately 150 different naturally occurring carotenoids are known. The structures of about 75 of these have been quite well established. The structural elucidations carried out before 1948 have been reviewed in detail 159

P r o g r e s s in the C h e m i s t r y o f F a t s a n d o t h e r Lipids

by Karrer and Jucker ls9 in their comprehensive monograph on carotenoids (revised edition in English translation of 1950).~9° In the following section the more recent structural studies reported after the 4'

2r

3%..51

-

-

l

1

y/<.,,

4

appearance of Karrer and Jucker's book will be discussed. Reference to carotenoids with previously well-established structures will only be made when necessary for the discussion. The numbering of the carbon skeleton, recommended by the American Chemical Society, 5 will be employed. A. Carotenes Carotenes are considered C40 hydrocarbons, chemically or biochemically related to lycopene, regardless of colour. The biological precursors of the coloured carotenoids, viz. phytoene, phytofluene and ~-carotene are often referred to as colourless or more saturated carotenes. The traditional grouping of carotenes into aliphatic, monocyclic and bicyclic compounds is maintained in this presentation, although the recently described aryl-carotenes, containing aromatic end-groups with rearranged carbon skeleton, might justify a different way of grouping. One knows at present eighteen naturally occurring carotenes (Tables 5 and 6). Seven are aliphatic, five monocyclic and six bicyclic representatives. Four of these carotenes have aromatic end-groups of the 1,2,5- or 1,2,3-tri-methylsubstituted type. 1. Aliphatic carotenes The aliphatic carotenes can formally be derived from lycopene (V) by partial hydrogenation or dehydrogenation. A characteristic feature of the more saturated carotenes is the presence of isolated double bonds, in agreement with the formal composition from 8 isoprene units. Phytoene (I). Phytoene is a non-crystalline oil, the structure of which was established by Rabourn and Quackenbusch. 274 The structural assignment has been confirmed by n.m.r.-spectroscopy and total synthesis carried out by Davis, Jackman, Siddons and Weedon. 74 Phytofluene (II). Rabourn and Quackenbusch~75 assigned the structure (II) to phytofluene also and the assignment was subsequently proved by n.m.r.spectroscopy and total synthesis, by Weedon and his associates. 74 ~-Carotene (III). Preliminary investigations by Nash, Quackenbusch and 160

Recent Progress in CarotenoidChemistry Table 5. Naturally occurring aliphatic carotenes ReferDesignation ence to structure

Structure

Phytoene

••

274,74

i

(~)

Phytofluene 275,74

{rrr) ~-Carotene

272,74

Neurosporene

74

Lycopene

190

(3ZZ) 3,4-Dehydrolycopene 193

~

3,4,3',4'Dehydrolycopene

(vl-r)

193,317

I

Porter 255 indicated a relatively saturated structure for this carotenoid. Degradation studies performed by the workers at Purdue suggested the symmetrical structure (III) for ~-carotene. 272 This was confirmed by total synthesis by Davis, Jackman, Siddons and Weedon. 74 The natural occurrence also of the isomeric, non-symmetrical compound with the conjugated double bond chain located between C-atoms 5 and 13' is anticipated. Neurosporene (IV), first described by Haxo, TM was for a period considered to 161

Progress in the Chemistry of Fats and other Lipids

Table 6--Part I. Naturally occurring monocyclic and bicyclic carotenes ReJbrStructure

Designation

ence to slrttClllre

(-~-m-~

/3-Zeacarotene

264, 280, 333

('7"I"F b )

a-Zeacarotene

264,333

(Ix)

y-Carotene

190

(x)

Torulene

280

3-Carotene

175,141

Chlorobactene

237, 24

(zI)

be identical with 6,7,6',7'-tetrahydrolycopene, synthesized by Eugster, Linnet, Trivedi and KarrerP 0 However, the result of the N-bromosuccinimide dehydrogenation of C-carotene to neurosporene, reported by Zechmeister and Koe,z~4 was not consistent with this formulation. The problem was elegantly solved by means of n.m.r.-data and by total synthesis, carried out by Davis, Jackman, Siddons and Weedon, 74 who established the structure (IV) for neurosporene. Flavorhodin, isolated from rhodovibrio-bacteria by Karrer and Solmssen,194 is probably identical with neurosporene. This might also be true for the so-called sarcinene obtained by Chargaff and Dieryckza, a9 from Sarcina lutea. 3,4-Dehydrolycopene (VI). Wintersteins38 has recently claimed that this carotenoid, obtained by Karrer and Rutschmann 19a upon dehydrogenation of lycopene by the action of N-bromosuccinimide, being naturally in oranges. 162

Recent Progress in CarotenoidChemistry Table 6--Part H

Structure

Designation

Reference to structure

#-Carotene

190

a-Carotene

190

k-Carotene

179, 37

Isorenieratene= leprotene

345, 240

Renieratene 345

(xvl

i~ )

Renierapur- 345 purin

3,4,3",4"-Dehydrolycopene (VII) has also been found by Wintersteinaa6 to be naturally occurring. This compound is obtained by N-bromosuccinimide treatment of lycopene,lgz and was recently obtained in high yield by Surmatis and Ofner317 through total synthesis. 2. Monocyclic carotenes

Monocyclic carotenes can be considered as derivatives of v-carotene (IX). The known members are listed in Table 6. ~-Zeacarotene (VIII), isolated from corn, is a provitamin of vitamin A and was characterized by Petzold, Quackenbusch and McQuistan. 264 N-bromosuccinimide treatment yielded v-carotene, and the structure as a 7",8'-dihydro163

Progress in the Chemistry of Fats and other Lipids 9,-carotene (VIII) was tentatively suggested by these authors. 7',8'-Dihydro-ycarotene was later synthesized by Riiegg, Schwieter, Ryser, Schudel and Islet. 2s° The properties of the synthetic specimen agreed quite well with those of natural fi-zeacarotene, although a direct comparison could not be carried out. 7',8'-Dihydro-7-carotene (VIII) as well as 7,8-dehydro-y-carotene has recently been synthesized by Weedon and his associates. 333 Direct comparison with a natural specimen secured the structure (VIII) for fl-zeacarotene. a-Zeacarotene (VIIIb) was also characterized by Petzold, Quackenbusch and McQuistan. 264 Based on lack of vitamin A activity, spectral data and the production of a compound indistinguishable from g-carotene (V1) on N-bromosuccinimide treatment, the structure as a 7',8'-dihydro-~-carotene was suggested. Weedon and his associates 333 have lately performed a total synthesis of (VIIIb). The properties were in agreement with those reported for natural a-zeacarotene. Torulene (X) is a characteristic carotene of red yeasts, 99 suggested by Lederer216 to be a monocyclic spirilloxanthin, and by Winterstein 336 to be a 3",4'-dehydroy-carotene. The latter compound was synthesized by Rfiegg, Schwieter, Ryser, Schudel and Isler 2s° and proved to be identical with torulene. g-Carotene (XI), first described by Wintersteiny 5 has recently been isolated from a particular variety of tomato. 175 This carotene is the a-ionone analogue of y-carotene (IX) and has no vitamin A activity. Thorough structural studies carried out by Kargl and Quackenbusch lw established the molecular composition of the compound. The presence of 12 double bonds in the molecule was demonstrated, and these were supposed to be conjugated to account for the absorption spectrum in visible light. Alkali isomerization, in forcing conditions, afforded y-carotene. The infrared spectra of perhydro-g-carotene and perhydro-ycarotene were identical. Ozonization studies also supported the structure (X1) for g-carotene. The structure (XI) was later confirmed by total synthesis carried out by the Hoffmann-La Roche group. 141 Chlorobactene (XII) is the major carotenoid of green photosynthetic bacteria, ez7 This carotene shows much resemblance to y-carotene (IX) in physical properties. However, n.m.r.-data clearly revealed the presence of an aromatic end-group. Its structure was elucidated by Liaaen-Jensen, Hegge and Jackman, 237 and subsequently confirmed by total synthesis performed by Bonnett, Spark and Weedon. 24 3. Bicyclic carotenes

Bicyclic carotenes are usually considered as derivatives of/3-carotene (XIII). Chemical structures for the six known members are presented in Table 6. The aryl-derivatives can formally be considered as having arisen from carotenoids with the usual 1,1,3-trimethyl substituted cyclohexene ring by dehydrogenation accompanied by Wagner rearrangements. In addition to the very abundant and widely distributed/3-carotene and a164

Recent Progress in CarotenoidChemistry carotene, ~-carotene and three aromatic representatives have recently been isolated from natural sources. c-Carotene (XV). This designation was given to a carotene first isolated from Navicula torquatum by Strain and Manning.811 The racemic mixture or mesoform of (XV) was synthesized by Karrer and Eugster179 and called El-carotene. Later, a synthesis of the optically active isomers of (XV) was reported by Tscharner, Eugster and Karrer. 324 Recently, Chapman and Haxo 37 have compared natural E-carotene from various algae with synthetic ~l-carotene of Karrer, and proved their identity. Isorenieratene (XVI); synonym leprotene. Yamaguchi's340-345 excellent work on the carotenoid components of the sea sponge, Reniera japonica, led to the discovery of a new class of natural carotenoids with aryl end-groups. The structure of isorenieratene (XVI) was first established by degradation studies 340,342 and subsequently proved by total synthesis by the same author 345 from oct-4-ene-2,7-dione and aryl analogues of/3-ionone. A similar synthesis was later reported by Khosla and Karrer. 199 Cooper, Davis and Weedon53 greatly improved the yield in a different synthetic route from crocetindial, employing the Wittig reaction. Liaaen-Jensen and Weedon233, 24o have recently shown that leprotene, first isolated by Grundmann and Takeda, 121 is identical with isorenieratene (XVI). Renieratene (XVII), from Reniera japonica has a non-symmetrical structure with one 1,2,5- and one 1,2,3-trimethyl-phenyl end-group. The structure was established by Yamaguchi340, 341,343 and proved by total synthesis performed by Yamaguchi.345 Better yield was obtained by Cooper, Davis and Weedon53 using the same approach as in their synthesis of isorenieratene. Renierapurpurin (XVII|), has two 1,2,3-trimethylphenyl end-groups. Renierapurpurin was characterized and later synthesized by Yamaguchi.340, 345 Again the synthesis has been improved by Weedon and his associates. 53

B. Carotenoids with hydroxy and methoxy groups only For the sake of convenience, the xanthophylls (oxygen-bearing carotenoids) will be divided into those carrying hydroxy and methoxy groups only, and those which contain carbonyl functions (ketones, aldehydes, carboxylic acids and esters) solely or together with hydroxy and methoxy groups. A number of new xanthophylls have been described during the last decennium, several of which have been isolated from photosynthetic bacteria. A characteristic structural feature of the latter type of xanthophylls is their aliphatic and frequently nonsymmetric structure, containing tertiary hydroxyl substituents located in 1,1'-positions. Occasionally these xanthophylls are methoxylated in these positions.

1. Aliphatic hydroxy and methoxy carotenoids The chemical structures of the new xanthophylls, to be discussed below, are 165

Progress in the Chemistry of Fats and other Lipids presented in Table 7. Except for a-bacterioruberin, which is the major carotenoid of certain obligatory halophilic bacteria, 16 these carotenoids are typical of photosynthetic purple bacteria. Concerning their distribution, the reader is referred to previous work by Goodwin 1°4, 106 and a more recent survey by Liaaen-Jensen. 232 The individual carotenoids will be discussed in the order of increasing length of the conjugated polyene chain. Chloroxanthin (XIX) has been characterized by Nakayama. 253 N.m.r. data Table 7--Part I. N e w aliphatie hydroxy and methoxy earotenoids ReferDesignation i ence to struclure

Structure

H H

3

i iI

O C

O

~

(XX l I) i

~

~

~

/

(~

Chloroxan- 253, thin i 227, 74, !333 Spheroidene 113,74 J

)

I i

OH230, 164 Spheroidene

Rhodopin

H0 H

~

~

~

0H(R-XTTT

~O ~ ~ -~ ~ ~~ ~ ~ ~

1,2,1',2'Tetrahydro1,1'-dihydroxyl lycopene

~-/ (XXlV)3,4-Dehy-

227, 281,24

317,[281

163

i dro-rhodopin 1 I

H3C 0

_

.

~

~

( &,~x,~iT)

166

Anhydrorhodovibrin

227

Recent Progress in Carotenoid Chemistry reported by Davis, Jackman, Siddons and Weedon 74 indicated that chloroxanthin was a hydrated neurosporene (XIX), a structure favoured by LiaaenJensen, 227 based on biosynthetic consideration and physical properties. The structure (XIX) has been proved beyond doubt by total synthesis, recently performed by Weedon and his associates. ~3 Spheroidene (XX), previously called Pigment Yellow, was first described by van Niel. 326 Goodwin, Land and Sissins na ascribed a chemical structure to it based on limited evidence. The analytical data obtained by Goodwin and collaborators were in essence confirmed by Nakayama. 254 However, the structure (XX) for spheroidene was unequivocally established by Davis, Jackman, Siddons and Weedon 74 by means of n.m.r.-spectroscopy. The name spheroidene was suggested by Shneour. zg° OH-spheroidene (OH--Y) (XXI). This preliminary designation was introduced by Eimhjellen and Liaaen-Jensen82 to indicate its relationship with spheroidene. Structural data obtained by Liaaen-Jensen23° were consistent with the structure (XXI) for OH-spheroidene. Confirmation was procured by the chemical dehydration to spheroidene (XX), and the n.m.r, data reported by Jackman and Liaaen-Jensen.1~

Table 7.--Part H Designation

Structure

Reference to structure

H3CO

OH

H3CO

OH(3t'3EEI'T)

Rhodovibrin

227

Monodemethylated spirilloxanthin

227,230

Spirilloxan- 14, 227, thin 317

H

O

~

o

H ("YY'I"W)

167

a-Bacterioruberin

227

Progress in the Chemistry of Fats and other Lipids

Rhodopin (XXII). This monohydroxy-carotenoid with lycopene chromophore was chemically well characterized already in the 1935-40 period by Karrer, Sohnssen and Koenig, TM, 196,197, 201 although no structure was suggested for the compound. Goodwin and Land 112 claimed identity of rhodopin with lycoxanthin (3-hydroxy-lycopene)--a claim disproved by Liaaen-Jensen, 222, 223, z2v who suggested a revised formula (XXII). Confirmation of this was later obtained from n.m.r.-data discussed by Ryvarden and Liaaen-Jensen, TM and by the chemical dehydration to lycopene (V) upon treatment with POCI3 in pyridine, as reported by Liaaen-Jensen, Hegge and Jackman. 23v Rhodopin has recently been synthesized by Bonnett, Spark and Weedon. 24 The synthetic specimen was in all respects identical with authentic rhodopin. Rhodopin was the first of a series of bacterial carotenoids shown to carry tertiary hydroxyl groups in l-positions. 1,2,1 ',2'-Tetrahydro- 1,1 '-dihydroxy-lycopene (XXIII) is the dihydroxy analogue of rhodopin (XXII). This compound became available through the synthesis of Surmatis and Of net 31v by condensation of crocetin dialdehyde and 7-hydroxy3,7-dimethyl-2-octenyltriphenylphosphoniumbromide. The addition of water to the isopropylidene group to form the hydroxylated end-group proceeded with high yield. The synthetic specimen was found by Ryvarden and Liaaen-Jensen 'zsl to be identical with a minor carotenoid present in Rhodomicrobium ~:annielii. It is presumably also identical with a minor carotenoid present in other photosynthetic bacteria. 2')v, ZSl 3,4-Dehydro-rhodopin (XXIV). Visible light absorption data and n.m.r. evidence, reported by Jackman and Liaaen-Jensen 163 unequivocally established the structure for this carotenoid. The properties of this carotenoid are very similar to those of rhodovibrin (XXVI). A nhydro-rhodolqbrin (XXV); synonym P481. Structural work has been carried out by Liaaen-Jensen, 2e0, '~_1,22v who suggested the structure (XXV) for anhydrorhodovibrin. There is some uncertainty about the exact localization of the chromophore in the molecule. The twelve conjugated double bonds may occur in a symmetric or a non-symmetric position. Biochemical arguments favour the structure (XXV). Rhodo~,ibrin (XXVI); synonym OH-P481. By considering previous data published by Karrer, Solmssen and Koenig 195-a97 and additional evidence, the structure (XXVI) was suggested by Liaaen-Jensen. 22°, 224, z2v As in the case of anhydro-rhodovibrin (XXV), the position suggested for the chromophore was based on biochemical arguments. Monodemethylated spirilloxanthin (XXVII) ; synonym OH-spirilloxanthin, is a trace carotenoid present in several photosynthetic bacteria, shown by LiaaenJensen'~7, 2~s to be identical with a-bacterioruberin monomethylether. Infrared evidence2~0 is in agreement with this formulation. Spirilloxanthin (XXVIII); synonym rhodoviolascin, was chemically characterized by van Niel and Smith 3zs and Karrer, Solmssen and Koenig. 194-197 The aliphatic nature of the molecule was established by oxidative degradation studies, 168

Recent Progress in CarotenoidChemistry and the presence of two methoxyl groups was demonstrated for the first time in a carotenoid. Based on the result of the isopropylidene determination and on absorption properties in the infrared region, the structure (XXVIII) was suggested for spirilloxanthin by Liaaen-Jensen.221, 227 Shortly afterwards Barber, Jackman and Weedon 14 published the result of a n.m.r, study of spirilloxanthin and also assigned the structure (XXVIII) to this carotenoid. A partial synthesis in low yield of spirilloxanthin by methylation of abacterioruberin (XX1X) has been reported by Liaaen-Jensen.221, z27 Recently Surmatis and Ofner317 have carried out an elegant total synthesis of spirilloxanthin. The synthetic sample was found to be identical with natural spirilloxanthin. a-Bacterioruberin (XXIX). This carotenoid was first described by Petter. 261, z62 Lederer217 pointed out the spectral resemblance of a-bacterioruberin and spirilloxanthin. Chemical studies were carried out by Liaaen-Jensen,225-227 who assigned the structure (XXIX) to a-bacterioruberin, a decisive argument being that monodemethylated spirilloxanthin and spirilloxanthin were obtained upon methylation in forcing condition. The all-trans isomer of a-bacterioruberin is exceptionally labile in solution. Spontaneous isomerization to cis-forms take place even in the dark, and the equilibrium mixture, after iodine catalysis in light, contains 63 per cent cis isomers.

2. Monocyclic hydroxy carotenoids The structural formula of recently described monocyclic as well as bicyclic representatives are depicted in Table 8. OH-ehlorobactene (XXX), is a monocyclic, aryl-xanthophyll present as a minor carotenoid in photosynthetic, green bacteria. Its structure was established by chemical dehydration to chlorobactene (XII), reported by Liaaen-Jensen, Hegge and Jackman237 and confirmed by total synthesis performed by Bonnett, Spark and Weedon.24 3. Bicyclic hydroxy carotenoids a-Cryptoxanthin (XXXI), 3'-hydroxy-a-carotene, from red pepper, has been described by Cholnoky, Szabolcs and Nagy.4s The visible light absorption spectrum, partition behaviour, adsorptive properties, the result of catalytic hydrogenation, formation of an acetate and biological vitamin A-activity all indicate that a-cryptoxanthin represents a mono-hydroxy-a-carotene with the hydroxy group in the 2' or 3'-position. Since a-cryptoxanthin occurred together with carotenoids hydroxylated in 3-position, the structure (XXXI) was preferred for this carotenoid. The designation fl-cryptoxanthin should now be used for cryptoxanthin (3-hydroxy-fi-carotene). The structure (XXXI) had previously been claimed for physoxanthin, isolated from Physalis alkekengi by Bodea and Nicoara2°. Their results were 169

Progress in the Chemistry of Fats and other Lipids Table 8. N e w monocyclic and bicyclic hydroxy carotenoids

Designation

Structure

ence to StrllCtllre

(wx'z)

OH-Chloro- 237,24 I bactene

(x'x~)

a-Cryptoi xanthin

48

i i

( xx-x-n-) i Zeinoxanthin

HO _

OH

(3r'~TW)

Eschscholtz- 192,16 xanthin

strongly criticized by Cholnoky et al. 48 who considered physoxanthin to be a cis-isomer of/3-cryptoxanthin. Zeinoxanthin (XXXII), from corn, has been characterized by Petzold and Quackenbusch. 263 Again the visible light absorption spectrum, partition ratio and adsorptive properties were taken as evidence for a mono-hydroxy-acarotene structure. This carotenoid was different from synthetic 4-hydroxy-acarotene, and the test for allylic hydroxyl groups was negative. Unlike acryptoxanthin (XXXI) zeinoxanthin was vitamin A non-active. The hydroxyl substituent is therefore located in the fl-ionone ring, and of the alternative positions 2 and 3, the latter was considered the most likely. The hydroxy-acarotene from oranges described by Curl 57 may be identical with zeinoxanthin (XXXII). Eschscholtzxanthin (XXXIII) and rhodoxanthin are the only known naturally occurring carotenoids with retro-structure. Eschscholtzxanthin was first examined by Strain, a°5 who established the molecular composition and the presence of twelve conjugated double bonds and two secondary hydroxyl groups in the molecule. The structure was elucidated by Karrer and Leumann, 192 who, on the basis of spectroscopic similarity with retrodehydro-fi-carotene, lack of 170

263

Recent Progressin CarotenoidChemistry acetone formation on ozonolysis, and conversion to the hydrocarbon anhydroeschscholtzxanthin by treatment with acid chloroform, assigned the structure (XXXIII) to eschscholtzxanthin. The latter reaction has since been frequently employed to establish the presence of allylic hydroxyl groups in carotenoids. A partial synthesis of eschscholtzxanthin by manganese dioxide oxidation of zeaxanthin (3,3'-di-hydroxy-/%carotene) has recently been reported by Jaeger and Karrer. 161 Table 9.--Part L New aliphatic and monocyclie carbonyl-containing carotenoids

Structure

[

~

CHO

~

C

H

O

(

(X'X'~'T~r)

~xxv)

(-x-~c~)

Designation

Reference to structure

Apo-8'lycopenal

338,24

Apo-6"lycopenal

1338,210

Spheroidenone

(~X~'STTT) OHSpheroide-

113,74

229,164

none

2,2'-Diketo- 164 spirilloxanthin (P518)

( x × x u t H)

0

~

~ ~

OCH3

CHO ( ~ )

CHO (XL)

171

I

Azafrinalde- 338 hyde

fl-Apo-10'carotenal (C~O

338,279

Progress in the Chemistry of Fats and other Lipids Table 9.--Part H Designa- Refertion ence to

Structure

SD~ttClltt'e

~ ~ ~

~

CHO

~

/

fi-Apo-8'- 338,279 carotenal (C,~o)

(]~L[)

C

HO (XLII)

CHO

(~XLTIT)

j3-Apo-2'- 338,279 carotenal (C~7)

3',4'338,279 Dehydro- ; 17'-oxo-7- !

carotene (C4o) COOH

(XLI~)

Torular- 143 hodin

C. Carbonyl-conta&ing carotenoids

This group comprises ketones, aldehydes, carboxyIic acids and esters. Aliphatic and monocyclic representatives with recently elucidated structures are listed in Table 9; new bicyclic members in Table 10. In the following, aliphatic, monocyclic and bicyclic carbonyl-containing carotenoids will be treated separately in the order of increasing length of the polyene chromophore, independent of the type of carbonyl function present. I. Aliphatic carbonyl-containing carotenoids Apo-8"-lycopenal (XXXIV); synonym apo-3-1ycopenal, was recently detected by Winterstein, Studer and Riiegg~8 in tomatoes, and was claimed to be identical with synthetic apo-8"-lycopenal. No experimental details concerning the preparation of the synthetic sample were reported. Apo-8'-lycopenal has recently been totally synthesized by ]3onnett, Spark and Weedon. z4 Apo-6"-lycopenal (XXXV); synonyms lycopenal, apo-2-1ycopenal, is again claimed by Winterstein, Studer and Rtieggz3s to be naturally occurring in tomatoes. The compound is obtained by chromic acid oxidation of lycopene (II) according to the method of Kuhn and Grundmann. zlo

172

Recent Progress in Carotenoid Chemistry Table 10. N e w bicyclic carbonyl-containing carotenoids Reference to structure

Designation

Structure

(~LV) 0

I i

Echinenone 102,332

0 Canthaxanthin

125,332

(XL-fZII) Capsanthin H0-~.../~1 '

I

86, 12

i

I OH

L

(XLW ' FR ' )" Capsorubin

o

(XLF~) Cryptocapsin

Spheroidenone (XXXVI); synonym Pigment Red. The occurrence is so far restricted to some aerobically grown Rhodopseudomonas spp. 100, 232 This carotenoid was first characterized by van Niel. 3z6 Goodwin, Land and Sissins uz established the presence of a conjugated carbonyl group, as well as a methoxy group in the molecule, and, despite insufficient evidence, assigned a structure to this carotenoid, which they renamed spheroidenone. Nakayama 254 considered his analytical data to be in accordance with the structure suggested by Gooawin et aL 11~ N.m.r. evidence presented by Davis, Jackman, Siddons and Weedon 74 N

173

86, 12, 54

45

Progress in the Chemistryof Fats and other Lipids unequivocally established structure (XXXVI) for spheroidenone, and required a considerable revision of the structure previously suggested by others. 11z The infrared spectrum shows complex absorption. The secondary alcohol obtained upon lithium aluminium hydride reduction of spheroidenone yielded 3,4dehydrolycopene on treatment with acid chloroform,z29 OH-spheroidenone (XXXVII); synonyms OH-R, hydroxy-Red.1°~, 229 This structure was suggested on the basis of structural studies carried out by LiaaenJensen2~9 and subsequently proved by n.m.r, data and chemical dehydration to spheroidenone (XXXVI) as reported by Jackman and Liaaen-Jensen.164 This preliminary designation is due to Eimhjellen and Liaaen-Jensen.s2 OH-spheroidenone is a characteristic carotenoid of certain, photosynthetic bacteria. 23'z 2,2"-Diketo-spirilloxanthin (XXXVIII); synonyms P512, l°° P518, 2zs exhibits absorption maxima at longer wavelengths than any other carotenoid hitherto described. It is a minor carotenoid found in some Rhodopseudomonas spp. grown under aerobic conditions. By considering the properties of the parent substance and the lithium aluminium hydride reduced compound, Liaaen-Jensen22s assumed the structure 2-keto-spirilloxanthin for P518. Based on n.m.r, data and its behaviour upon reduction with zinc-acetic acid-pyridine, the symmetrical structure as a 2,2'diketo derivative of spirilloxanthin was later established by Jackman and Liaaen-Jensen.164

2. Monocyclic carbonyl-containing carotenoids Azafrin-aldehyde (XXXIX). A new carotenoid aldehyde found in Escobedia scabr(folia is, according to Winterstein, Studer and Riiegg, 338 presumably identical with azafrin-aldehyde (XXXIX). This compound occurred together with azafrin, the structure of which has previously been firmly established by Kuhn and Deutsch. 2°9 [3-Apo-lO'-carotenal (CzT) (XL), is, according to Winterstein, Studer and Rfiegg,zz8 a minor, but rather widely distributed carotenoid aldehyde. This aldehyde is available through the elegant total synthesis of/3-carotenals carried out by Rtiegg, Montavon, Ryser, Saucy, Schwieter and Isler. 279 [3-Apo-8'-carotenal (Cz0) (XLI). Natural occurrence and synthesis has been reported as for/3-apo-10'-carotenal (XL) above. fi-Apo-2"-carotenal (C37) (XLII) has been found by Winterstein, Studer and Riiegg338 in citrus fruits. Again, authentic material for comparison was made available through the total synthesis of this aldehyde by Riiegg, Montavon, Ryser, Saucy, Schwieter, and Isler. z79 3",4"-Dehydro-17"-oxo-7-carotene (C40) (XLIII) has been reported by Winterstein, Studer and Rtieggms to be present in an unspecified type of microorganism, also producing torulene (X), and was found to be identical with the synthetic aldehyde (XLIII) of Rfiegg, Montavon, Ryser, Saucy, Schwieter and Isler. 27s Torularhodin (XLIV). The early history and chemistry of torularhodin has 174

Recent Progress in Carotenoid Chemistry been reviewed by Karrer and Jucker. 19° This acid carotenoid was formulated as /3-apo-2'-carotenoic acid (C37) by Karrer and Rutschmann. 19z By elegant synthesis of a vinylogous series of/3-apo-carotenoic acids, Islet, Guex, Rfiegg, Ryser, Saucy, Schwieter, Walter and Winterstein~43 were able to prove the structure (XLIV) for torularhodin as a C40-acid, in which one of the methyl substituents of the gem-dimethyl group had been oxidized to a carboxyl group.

3. Bicyclic carbonyl-containing carotenoids The chemical structures of new bicyclic carbonyl-containing carotenoids are listed in Table 10. Echinenone (XLV), 4-keto-/3-carotene, is possibly identical with myxoxanthin and aphanin. 19°, 102 The identity with aphanin has, however, been disputed by Tischer. 322 Previous structural work carried out with myxoxanthin and aphanin has been discussed by Karrer and Jucker. 19° This keto-carotenoid is apparently widely distributed in algae and marine vertebrates, according to the data collected by Goodwin. 1°2 Echinenone was first isolated by Lederer. 216 Goodwin and Taha 114 and Goodwin~07 carried out the reduction of echinenone to isocryptoxanthin (4-hydroxy/3-carotene), which could be dehydrated to retrodehydro-[3-carotene according to the procedure developed by Wallcave and Zechmeister.3a0 Echinenone, therefore, is 4-keto-/3-carotene (XLV). A partial synthesis of echinenone has been reported by Ganguly, Krinsky and Pinchard 99 by Oppenauer oxidation of isocryptoxanthin, produced by the action of boron trifluoride on retrodehydro-[3-carotene. Echinenone (XLV) was also obtained in low yield by Petracek and Zechmeister25s on treatment of fl-carotene with N-bromosuccinimide in alcohol-containing chloroform. A rational synthesis was developed by Warren and Weedon332 from fl-apo8'-carotenal (XLI) and 6-ethylenedioxy-3,3-dimethyl-octan-2-one to a diketone which, on alkali-catalyzed cyclization, gave echinenone (XLV). A different synthesis of echinenone by use of Robinson's Mannich base reaction for the formation of the cyclo-hexenone ring has since been reported by Akhtar and Weedon. 3 Canthaxanthin (XLVI), 2,2'-diketo-/3-carotene, was first isolated by Haxo ~25 from the mushroom Cantharellus cinnabarinus. It occurs also in other biological sources as bacteria282 and crustaceae. 321 Canthaxanthin (XLVI) was partially synthesized by Petracek and Zechmeister25s according to the method employed in the echinenone synthesis of the same authors. Warren and Weedon382 and Akhtar and Weedon3 have reported the total synthesis of canthaxanthin using the same principles as in their echinenone synthesis referred to above. Zeller, Bader, Lindlar, Montavon, Miiller, Rtiegg, Ryser, Saucy, Schaeren, Schwieter, Stricker, Tamm, Zfircher and Isler857 have synthesized canthaxanthin according to the C19 (dehydro-retro-C19-aldehyde) + C2 + C19 principle. The oxygen 175

Progress in the Chemistryof Fats and other Lipids functions were introduced as acetoxy groups, subsequently saponified and oxidized (Oppenauer oxidation) to conjugated keto-groups. Capsanthin (XLVII). The chemical structures of the major paprika ketones, capsanthin and capsorubin, has been a major problem in carotenoid chemistry for a long period, successfully solved during recent years. Chemical investigations carried out during the early period by Zechmeister and Cholnoky, and Karrer and collaborators have been summarized by Karrer and Jucker 19° and Cholnoky, Szab6 and Szabolcs. 4n As a result of these investigations capsanthin was formulated as a monocyclic keto-carotenoid (XLVIIa). On the basis of changes in the carotenoid composition of paprika fruits during ripening, Cholnoky, Gy/Srgyfy, Nagy and Pfincz6144 later advanced the hypothesis that antheraxanthin (zeaxanthin-5,6-epoxide) was the biosynthetic precursor of capsanthin. Entschel, Eugster and Karrer ss subsequently assigned the structure (XLVIIb) to capsanthin. 0

OH (XL~[a)

HO

0~

OH

(XL3ZII: b ) H0 ~..~,,,,~ •

i

I

(XL-IZ~c) HO

/

/

-°"

~

~<~

(X L ¥ ] Z )

H

Cholnoky, Szab6 and Szabolcs 46 reported the elementary analysis of a number of synthetic capsanthin esters and revised the molecular formula to C40H5603. Basing their results on the spectral properties of the product obtained by treatment of capsorubol with acid chloroform, Cholnoky and Szabolcs 47 assigned the structure (XLVIIc) to capsorubin, although the isopropylidene value obtained by Cholnoky, Gyt~rgyfy, Nagy and P~ncz6144 was not consistent with this formulation. Hydrogenation data by Entschel and Karrer 86 subsequently proved that capsanthin contained ten carbon-carbon double bonds only. Oppenauer oxidation of capsanthin gave a hydroxy-diketone, capsanthon, with additional infrared absorption at 5-75/z, typical for a 5-ring ketone. Ozonolysis of capsanthin gave a trimethyl-cyclopentanol-carboxylicacid. Structure (XLVII) was assigned 176

Recent Progress in Carotenoid Chemistry to capsanthin. Shortly afterwards, Barber, Jackman, Warren and Weedon12 presented independent support for the structure (XLVII) for capsanthin, including n.m.r.-evidence. The methyl groups of the cyclopentane ring exhibited signals at 9.16, 8.80 and 8.63 ~-. Faigle and Karrer 92, 93 later considered the stereochemistry of capsanthin and claimed a cis-relationship of the oxygen substituents on the cyclopentane-ring. By synthesis work, Cooper, Jackman and Weedon54 arrived at the opposite conclusion. Capsorubin (XLVIII). The elucidation of the structure of capsorubin parallels that of capsanthin (XLVII), and the reader is therefore referred to the literature cited under capsanthin for the major references. It is a ~o,o;-conjugated diketone with two cyclopentane end-groups of the same type as in capsanthin (XLVII). Cooper, Jackman and Weedon54 have recently carried out a total synthesis of capsorubin. Cryptocapsin (XLIX) was first described by Cholnoky, GySrgyfy, Nagy and P~incz6144 as a minor carotenoid component of red pepper. Following the elucidation of the structures of capsanthin and capsorubin, the structure (XLIX was proposed by Barber, Jackman, Warren and Weedon,12 and subsequently proved in an elegant manner by Cholnoky, Szabolcs, Cooper and Weedon,45 by preparation of the same type of derivatives which proved to be of decisive importance for the establishment of the capsanthin and capsorubin structures.

D. Epoxidic andfuranoid carotenoids The establishment of the structures of this class of carotenoid pigments was the brilliant achievement of Karrer's school in 1945-50. The so-called epoxidic carotenoids have ether bridges between carbon atoms 5 and 6, whereas the furanoid rearrangement products obtained on acid treatment have oxygen bridges between carbon atoms 5 and 8. The designation "5,8-epoxide" t0t the furanoid oxides 1°2, 6z should be avoided, since such 5,8-oxides are not real epoxides. Some of the furanoid oxides are naturally occurring. The epoxides and furanoid oxides with previously known structures are derivatives of fl-carotene, zeaxanthin~ a-carotene and lutein, as is shown in Table 11. In addition, some of the carotenoid epoxides, first obtained by synthesis have been found in nature, and the structure of one more carotenoid belonging to this class has been established. The chemical structures of these carotenoids are depicted in Table 12. Furthermore the isolation of a number of new carotenoids of this type has been reported, whose structures are not yet determined (see the following section). Epoxidic and furanoid carotenoids are found in abundance in fruits and flowers, as well as in green plants and algae. Cryptoxanthin monoepoxide (L) was obtained by Karrer and Jucker 188 by monoperphthalic acid oxidation of cryptoxanthin (3-hydroxy-fl-carotene). Since the synthetic compound was Vitamin A inactive it contains no uns~:bstituted fl-ionone ring. The furanoid rearrangement product was given the name 177

Progress in the Chemistry of Fats and other Lipids Table 11. Epoxidic and furanoid derivatives of fl-carotene, zeaxanthin, a-carotene and lutein 19° Chemical structure

Designation 5,6-Epoxide

fl-Carotene-monoepoxide fl-Carotene-diepoxide Luteochrome Flavochrome Antheraxanthin Violaxanthin Mutatoxanthin=Citroxanthin Auroxanthin a-Carotene-expoide Flavochrome Luteinepoxide=Eleoxanthin Flavoxanthin } Chrysanthemaxanthin

~

Furanoid oxide

/3-carotene-monoepoxide fl-carotene-diepoxide /3-carotene-monoepoxide-monofuranoid-oxide /3-carotene-difuranoid-oxide zeaxanthin-monoepoxide zeaxanthin-diepoxide zeaxanthin-monofuranoidoxide zeaxanthin-difuranoid-oxide a-carotene-epoxide a-carotene-furanoid-oxide lutein-epoxide lutein-furanoid oxide

~

R

5,6 -epoxide

R

Furanoid oxide

cryptoflavin. A carotenoid with properties similar to cryptoxanthin monoepoxide has recently been found in lemons and prunes by Curl. 62, 65 Cryptoxanthin diepoxide (LI). The partial synthesis was performed by Karrer and Jucked 88 in the same way as for the monoepoxide (L) above. The difuranoid rearrangement product is called cryptochrome38s Cryptoxanthin diepoxide also appears to be naturally occurring in lemons and prunes according to Curl. n2, 6.~ Trollixanthin (LI1). The carotenoid epoxide, trollixanthin, was first isolated from the flowers of Trollius europaeus by Karrer and Jucker; 186 molecular formula C40H5604.186, 191 The absorption spectrum in visible light corresponds to that of luteinepoxide, whereas the spectrum of the furanoid rearrangement product, trollichrome, is similar to that of crysanthemaxanthin and flavoxanthin. Karrer and Krause-Voith, TM therefore, assumed troUixanthin to be a derivative of lutein-epoxide with one additional hydroxyl substituent. The structure (L]I) is due to Lippert, Eugster and Karrer. 243 Catalytic hydrogenation revealed the presence of ten carbon-carbon double bonds. Trollixanthin was not affected by either periodic acid or lead tetraacetate treatment and 178

Recent Progress in Carotenoid Chemistry

Table 12. New carotenoid epoxides and furanoid oxides ReferStructure

HO

Des~natmn

ence to structure

Cryptoxanthin monoepoxide

188, 62

Cryptoxan-

188, 62

thin diepoxide

HO HO

OH (LII)

Trollixanthin

243

HO

therefore possessed no e-glycol arrangement. Perhydro-trollichrome gave no triacetate, but afforded a diacetate, which, on saponification, eliminated water and gave anhydro-perhydro-trollichrome. This evidence indicated the presence of a tertiary hydroxyl group in trollichrome and trollixanthin. The structure (LII) was ascribed to the latter carotenoid; trollichrome being (LIIa).

~

OH

(L~al

HO

E. Carotenoids with partly known structures During the last fifteen years a number of new carotenoids have been described, the structures of which are not yet known in detail. Additional information concerning the structures of previously characterized carotenoids with unknown or partly known structures has also become available. In the following an attempt will be made to give key references to new carotenoids sufficiently characterized to be named individually, as well as to new evidence of importance for the establishment of structural formulae for previously described carotenoids. Carotenes, hydroxylated carotenoids, carbonyl-containing carotenoids and epoxidic-furanoid carotenoids will be treated separately. Within each class those compounds fm which structural formulae have been suggested will be discussed first. 179

Progress in the Chemistry of Fats and other Lipids

1. Carotenes v-Carotene, has not yet been obtained in pure state. It was first isolated by Goodwin 103 from berries of Lonicera japonica. Its absorption spectrum in visible light is similar to that of ~-carotene (IV). This compound, however, exhibits weaker adsorptive properties, and the structure (LIII) as a bicyclic, ~-carotene was tentatively suggested by Rabourn. 273

(L]~.)

2. Hydroxylated carotenoids Myxoxanthophyll, C40H5607. The early literature has been reviewed by Karrer and Jucker. 19° The tentative structure (LIV) proposed by Karrer and Rutschmann does not seem to be in agreement with the properties of this xanthophyll, although the criticism raised by Goodwin x02 is not appropriate. Goodwin 108 claims that a conjugated carbonyl group is not present. 0

(L'I~T) HO

OH

There is a controversy about a possible identity of myxoxanthophyll and aphanizophyll. 1°8 Hertzberg 1~8 has recently carried out a paper-chromatographic comparison of the carotenoids, of Oscillatoria rubescens (original source of myxoxanthophyll) and Aphanizomenon flos-aque (original source of aphanizophyll). The latter contained two carotenoids with absorption spectra similar to that of myxoxanthophyll of which the major one could not be separated from myxoxanthophyll on kieselguhr-containing paper. These two carotenoids appear not to be cis-trans isomers. Phlei-xanthophyll. A carotenoid similar to myxoxanthophyll was reported by Schlege1285 to be present in a strain of Mycobacterium phlei. However, this carotenoid is chromatographically different from myxoxanthophyll. Phleixanthophyll contains no carbonyl or methoxy groups and is a strongly polar xanthophyllJ ~9 180

Recent Progress in Carotenoid Chemistry

Hydroxy-phytoene. A compound with properties predicted for a monohydroxy-phytoene has been found in lemons by Curl. 62 Phytofluenol from ripe tomatoes, reported by Zechmeister and Pinchard,z~5 is presumably a hydroxy derivative of phytofluene (II). A mono-hydroxyderivative of phytofluene has also been found by Curl 62 in lemons. Also, from cultures of Rhodospirillum rubrum grown in the presence of diphenylamine, a similar compound has been isolated.235 The two latter xanthophylls are not necessarily identical with phytofluenol. Hydroxy-~-carotene. Such a compound has been found by Curl 62, 60 in apricots and lemons, and a compound with the predicted properties is also present in cultures of Rsp. rubrum grown in the presence of diphenylamine.235 Again the position of the hydroxyl group is unknown. Warmingol (Pigment 3) isolated from the sulphur purple bacterium Chromatium warmingii has been characterized by Liaaen-Jensen and Schmidt.239 It exhibits absorption properties in visible light similar to that of lycopene, contains one tertiary and one secondary, allylic hydroxy group and no methoxy groups. TM Moreover, warmingol is obtained on lithium aluminium hydxide reduction of warmingone (see below). Diatoxanthin, first isolated by Strain, Manning and Harding312 from diatoms is a xanthophyll very similar to zeaxanthin. It is not a cis-isomer of zeaxanthin, and it does not seem to contain any allylic hydroxy group. 172 3. Carbonyl-containing carotenoids Hydroxy-echinenone. The tentative structure (LV) as an a-ketol was assigned to a carotenoid isolated from Euglena gracilis by Krinsky and Goldsmith.2°5

(L~)

0

Euglenanone, was found by the same authors in the same source. The structure (LVI) was proposed for euglenanone. By analogy with the transformation of astaxanthin to astacin, reported by Kuhn and Sorensen,211 one would expect hydroxy-echinenone to yield euglenanone on saponification in the presence of oxygen, if the postulated structures were correct.

o 181

Progress in the Chemistry of Fats and other Lipids

Asteroidenone, from starfish, has been tentatively formulated as 3-hydroxy-4'keto-fi-carotene (LVII) by Nicola. 256 O

(LSZZ) HO Hydroxy-asteroidenone, assumed to possess structure (LVIII), was also isolated by Nicola 256 from the same source.

~

OH

HO

0

Asterin acid, assumed by Karrer and Jucker 190 to be identical with astacene (3,Y-4,4'-tetraketo-/3-carotene) has been re-isolated from starfish. Visible light

H

3

0

C

O

~

o

H (L]3Co)

and infrared absorption data, chromatographic behaviour and melting point indicate identity with astaxanthin (3,3'-dihydroxy-4,4'-diketo-fl-carotene).TM "2-Keto-rhodovibrin". A trace carotenoid from Rhodopseudomonas gelatinosa was given the structure (LIX) by Liaaen-Jensen. 231 After the revision of the P518-structure to 2,2'-diketo-spirilloxanthin by Jackman and Liaaen-Jensen, TM the above conclusion is less obvious, also bringing structure (LIXa) into question for this minor carotenoid.

Tangeraxanthin, obtained from tangerines by Curl, 63 was assigned the speculative structure (LX).

HO

~

CH3) 182

(LX)

Recent Progress in Carotenoid Chemistry

Reticulataxanthin was isolated from Citrus reticulata by Curl and Bailey, 69, ~ who ascribed to it the very tentative structure (XLI).

HO

Minor paprika ketones. In addition to capsanthin (LXVII), capsorubin (XLVIII) and cryptocapsin (LXIX) Curl 61 has described four other minor keto-carotenoids in red bell pepper. Warmingone, from the photosynthetic bacterium Chromatium warmingii, presumably contains eleven double bonds in conjugation with a keto group, za9 A tertiary hydroxyl group, but no methoxyl group is present. 234 Anhydro-warmingone, Pigment 1, from the same bacterium, has lately been obtained by chemical dehydration of warmingone. 2a4 Okenone, occurrence so far restricted to Chromatium okenii, was characterized by Schmidt, Liaaen-Jensen and Schlegel. 286 It represents a new type of ketocarotenoid, and possesses one methoxylated and one aryl end-group. 2as Fucoxanthin is the carotenoid characteristic of brown algae and of diatoms and is one of the last major carotenoids still lacking a molecular formula. Early work has been summarized by Karrer and Jucker. 190 Working on infrared evidence, Liaaen and Sorensen 219 and, independently, Torto and Weedon 323 realized the presence of an unusual allene grouping and two different carbonyl functions in fucoxanthin. Jensen 168 prepared a monoacetate, and, on treatment with lithium aluminium hydride, obtained the reduction products semifucoxanthol and the fucoxanthols. 169 The presence of an acetoxy group in fucoxanthin has recently been demonstrated by Jensen. 171 This new evidence indicates that the six oxygen functions of fucoxanthin are present as: one secondary hydroxyl group, two tertiary hydroxyl groups, one conjugated keto group and one acetoxy grouping. The chromophore apparently consists of eight conjugated double bonds terminated by a conjugated keto group on one side and an allene grouping on the opposite side. N.m.r. data seems to support a bicyclic structure. 172

4. Epoxidic and furanoid carotenoids Neoxanthin, C40H5604, a common carotenoid of green leaves, is found widely distributed, and was first characterized by Strain. 30a Curl and Bailey 67 demonstrated the epoxidic nature of neoxanthin and considered it as a triol monoepoxide. Goldsmith and Krinsky 115proposed the tentative structure (LXII) 183

Progress in the Chemistry of Fats and other Lipids for neoxanthin with the fourth oxygen atom as a tertiary hydroxyl group in 5' or 6'-position.

HO

(LXll) HO

Foliaxanthin, described by Cholnoky, Gy6rgyfy, Nagy and P~inc61,44 has been considered to be identical with neoxanthin. 6v, 115 Taraxanthin, C40H5604. The early literature has been discussed by Karrer and Jucker. 19° Eugster and Karrer 89 established the presence of a 5,6-epoxidegrouping in taraxanthin, which on acid treatment yielded the furanoid rearrangement product, tarachrome. This reaction had also been reported by Strain. z°8 Eugster and Karrer 89 consider taraxanthin to be a hydroxylated lutein-epoxide different from trollixanthin (LII). This suggestion is in agreement with the spectral properties of the product obtained by Jaeger and K a r r e d 61 on removal of the epoxidic oxygen in taraxanthin. Booth 28 recently reported that taraxanthin is present in dandelion fowers as a diester, taraxien, mainly a dipalmitate. The monoester also was found. Trolliflor, C40H5~O5, characterized by Lippert and Karrer, TM is an epoxidic carotenoid apparently closely related to trollixanthin (LII). On acid treatment the furnoid epoxide trolliflavin was obtained. Diadinoxanthin, is a lutein-like xanthophyll, first isolated from dinoflagellates by Strain, Manning and Harding. 312 It is not a c/s-isomer of lutein, m Krinsky 9-°4 has recently revealed the presence of a 5,6-epoxy grouping in diadinoxanthin. Direct comparison with zeaxanthin-monoepoxide might prove profitable. Persicaxanthin, a short-chromophore polyol 5,6-epoxide of undetermined structure has been described by Curl. 58 Valenciaxanthin, so named by Curl and Bailey, 6~ is a polyol 5,6-epoxide with about six conjugated double bonds, isolated from orange juice. Sinensiaxanthin was described by the same authors as a polyol 5,6-epoxide with somewhat longer chromophore. Luteoxanthin, also from orange juice, was tentatively identified as zeaxanthin monoepoxide-monofuranooxide by Curl and Bailey. 66 Luteoxanthin-like carotenoids were produced by citric acid treatment of violaxanthin (zeaxanthindiepoxide). Minor paprika epoxides. Curl 61 has claimed the presence of a number of minor carotenoids in red pepper, some of which presumably represent 5,6epoxides and furanoid oxides related to capsanthin (LXVII). 184

Recent Progress in Carotenoid Chemistry

IV. SYNTHESIS OF CAROTENOIDS

A. Total synthesis 1. General considerations The most brilliant achievement in the carotenoid field is undoubtedly the total synthesis of a large number of naturally occurring carotenoids by the schools of Inhoffen, Karrer and Weedon, and the research group of Islet. The first total synthesis of a carotenoid (fl-carotene) was carried out almost simultaneously by Karrer and Eugster, 17s Inhoffen, Bohlmann, Bertram, Rummert and Pommer1al and Milas, Davis, Beli6 and FlUs251 in 1950, following different routes. Since then about seventy carotenoids have been totally synthesized according to various principles. A detailed treatment of this excellent work is beyond the scope of the present survey, which will be restricted to a summary. For pertinent reviews in this particular field the reader is referred to articles by Isler, 140 Isler and Zeller,a59 Islet and Montavon,151 Isler, Rfiegg and Schudel,157 Pommer,266 Isler and Schudel, 15s as well as to the original work by Karrer's and Isler's groups, mainly presented in Helvetica Chimica Acta, and that of Weedon's group, reported in the Journal of the Chemical Society and the Proceedings of the Chemical Society, from 1950 onwards. In addition, partial synthesis has been reported for a number of carotenoids, in some cases starting with carotenoids which have been totally synthesized. The latter type can therefore be considered as totally synthesized. However, these conversions will be discussed in a separate section below.

2. Principal reactions employed Most carotenoids exhibit closely related skeletons with forty carbon atoms. Moreover, the coloured representatives normally have a common type of polyene chain in the central part of the molecule. General schemes have therefore been developed for the synthesis of coloured C40-carotenoids, following the schemes Cz + Cu + Cz = C40 or Cz -k Cu = C40, as very lucidly summarized by Islet and SchudeP5s (see Table 13). Three main reaction types have been employed for the total synthesis of the carotenoid skeleton. These are: (a) Grignard reactions of carbonyl compounds with acetylene magnesium halogenides according to the general scheme: R1

// Alkyl--C--C--MgX -+- O = C

\

Rz 185

R1

[

> Alkyl--C~C--C--R2 I OH

Progress in the Chemistry of Fats and other Lipids

Table 13. Principal schemes for total synthesis of carotenoids 1as Reaction type

Cx -4--Cy j Cx = C4o

C5 + C3o + C~

Grignard Grignard Wittig Grignard or Nef Wittig Grignard or Nef Enolether condensation Wittig Wittig Aldol condensation Robinson's Mannich base synthesis

Cx + Cy = C4o

Reaction type

Cv~ ~ C2

t C1~

C18+ C4 + C~8 C16 -- C8 --- C16 C15 @ Clo @ C15 C14 @ C12 ? C14 Cla + Cla

C13

Cao + C20 + C10

Enolether condensation Wittig Wittig Robinson's Mannich base synthesis Wittig Aldol condensation Wittig Grignard or Nef Grignard Wittig Wurtz (Reductive dimerization

C37 ~- C3 C'.~ ~ C5 C3o-i Clo C25 + C1~ C21 + C1~ C~o + C2o

(b) Wittig condensations o f c a r b o n y l c o m p o u n d s with triphenylalkylidenp h o s p h o r a n e s or d i a l k y l - a l k y l i d e n - p h o s p h o n a t e a n i o n s o f the f o l l o w i n g types:

R1

\

Ra

/

R1

\

C ~ P - - ( C 6 H 5 ) 3 + O~---C

/

>

R1

\ /

R2

/ R1

/ C~P

OH

I\~

O®OR

/ + O=C

R3

R4 186

\,

R2

R1

\

~

"\

/ R2

R3

C~C

\

R2

/

R4

/ C=C

R~

\ R4

Recent Progress in Carotenoid Chemistry (c) Enolether condensations. This procedure is not based on organometallic reagents, but employs the acid-catalyzed addition of acetals to enol ethers: OR

R2

{

R2

{

{

OR

/

R1--CH -k- C H ~ C H ----> R1--CH--C--CH

{

{

r

OR

OR

OR

H

\ OR

Other reactions less extensively used are: (d) Aldol-condensations; base-catalyzed condensations of two carbonylcompounds: R2

R8

{

R2

{

R3

{

{

R 1 - - C ~ O + C H s - - C = O ----+ R1--C--CH2--C~O

I

OH

(e) Wurtz-reactions with formation of hydrocarbons from alkyl iodides and alkyl-sodium: R1 1 q- RzNa ---+ Rt--R2 (f) Reformatsky synthesis, involving reaction between an a-halo-ester and activated zinc, followed by addition to aldehydes or ketones. This reaction was employed by Inhoffen and RaspO 37 for an intermediate in their bixin methyl ester synthesis:

~

CHO

OHC

|

H

+2 Br CHz-COOCH3

/

~

.

/

C

1

O

O

C

~

~

~

OH

cOOCH3

(g) Knoevenagel-Doebner condensation of aldehydes with compounds containing activated hydrogens, such as malonic acid, in the presence of pyridine and piperidine, as modified by Doebner. This reaction type is exemplified in the synthesis of bisnorcrocetin reported by Ahmad and Weedon :2 187

Progress in the Chemistry of Fats and other Lipids

OHC~ . ~ . ~ . ~ C H O

-F2 CH~(COOH,:

HOOC

cOOH

(h) Robinson's Mannich base synthesis, as employed by Akhtar and Weedon3 for the base-catalyzed condensation of a polyene ketone with the methiodide of a Mannich base, resulting in the formation of a cyclohexene derivative: 0

CH3C2H5

1® 0

°

R

~

~--

R

(i) Reductive dimer&ation with P2S5, as employed by Robesone77 for the synthesis of fl-carotene from Vitamin A aldehyde:

2~

C

H

O

I P2Ss

or via thiapyrane intermediates, as described by Chechak, Stern and Robeson. 4o The synthetic approach to the middle component and the ring components (see Table 13) has recently been thoroughly surveyed by Isler and Schudel. 15s Dehydrolinalool, citral and fl-ionone are used as starting materials in a number of carotenoid synthesis. A convenient synthesis of the very important intermediate C19-aldehyde is outlined in Fig. 10. By the method of Kimel, Surmatis, Weber Chase, Sax and Ofner,200 acetone and acetylene are condensed to a methylbutynol, which on partial hydrogenation affords methylbutenoL 188

Progress in the Chemistry of Fats and other Lipids

-- y . Acetone

Methytbut.enol

Methylheptenone

~Ac De h y d r o l i n a l o o I acetate

Dehydr olinalool

~

C HOAc ~

Citralallen-

~ C H O

Citral

ocetote

o -

PBeudoinone

~- Inone

G LYC f DE ST E RSY N T HE$ IS

~- C14 - Atdehyde

~

HO

- C16 - Aldehyde

VINYLETHERSY NTHESIS

~C~C,H,+\ ~AAAA,°C,H~, ~- C19" Aldehyde PROPENY LETHERSYNTHES IS

FIG. 10. Syn~.hesis c,f fl-CIg-aldehyde.

Methylheptenone is obtained by treatment of methylbutenyl-bromide with acetoacetic ester, and yields, on addition of acetylene dehydro-linalool, a key intermediate. The transformation of dehydro-linalool acetate to citral via citralallen acetate is due to Saucy, Marbet, Lindlar and Isler. ~84 Citral can also be extracted from lemongrass oil. By acetone condensation of citral pseudoionone is formed, and fl-ionone is obtained on acid catalyzed cyclization. The conversion of fl-ionone to fl-C14-aldehyde is performed by glycidic estersynthesis followed by alkali treatment, according to Islet, Huber, Ronco and Kofler. 146 The acetal is condensed with propenylether, using zinc chloride as

O

189

Progress in the Chemistry of Fats and other Lipids catalyst, and the intermediate yields fi-C14-aldehyde on acid treatment, fi-C14aldehyde is transformed in an analogous manner to fl-C19-aldehyde by propenylether-synthesis, as described by Islet, Lindlar, Montavon, Rtiegg and Zeller. x49 This exceedingly interesting reaction sequence with alternate vinylether and propenylether synthesis allows the gradual chain-lengthening of the isoprenoid carbon skeleton, fi-C19-aldehyde is a key intermediate in the synthesis of cyclic carotenoids, as may be seen from Fig. 1I, taken from the publication of Isler and Schudel. tSs Astocene

.j" Canthaxan~hin

~-eoroten~

~'15s15'- D e h y d r o - / I J~-c aro~ene t

Torulorhodln

O-c°.o~... "'~1s.~5-o.,y,~o~-Carotene

J~-Z . . . . . .

~ ~ene /

~-Opo-12'....

,o

Y 4- .y,",'o,,y-~-

~

~-Apo-

tenal (C25)

~

apocaPotenals

carotenats ~3,4-

~-Clg-AIdehyde

DehydPo-~-

opocaro~enals 3,4-Monodehydr o- '~/ °

..... ,.n.

3,4-Dehydro7, 7 " - d i h y d r o - capotene

/

/

\

7, 7 ' - Di h y d r o - caro~,ene

~

",,,

I~-Apo-carotenoic

.+,.r ~ -CaPo,ene

4,7 - Oi h y d r o dehydro - pert'o- c a r o t e n e

FIG. 11. fl-Cl~-aldehydeas intermediate for synthetic carotenoidsY~ As a detailed example of a carotenoid synthesis the technical synthesis is given in Fig. 12 of fl-carotene from fl-C19-aldehyde, the work of Isler, Lindlar, Montavon, R~iegg and Zeller¢ 49 Two moles of/3-C19-aldehyde is condensed with acetylene in a Grignard reaction. By allylic rearrangement and simultaneous dehydration of the C40-diol, followed by partial hydrogenation with Lindlar catalyst, 2+2 15,15'-cis-fl-carotene is formed. The latter compound is readily isomerized to all-trans-fi-carotene. A series of alternative routes has been developed for the synthesis of fl-carotene (for references see Table 14). The majority of totally synthesized C40-carotenoids are carotenes (hydrocarbons) of bicyclic, monocyclic or aliphatic nature. Concerning the total synthesis of oxygen-containing carotenoids, carotenoids with allylic oxygen functions in 4-positions are most readily available, but some carotenoids with oxygen functions in 3-positions also have been synthesized. Hydroxy groups in 4-positions can be introduced by an allylic rearrangement, as in the isozeaxanthin synthesis of Isler, Lindlar, Montavon, Riiegg and Zeller 150 or the polyene is treated with N-bromosuccinimide in acetic acid (allylic acetate groups introduced) according to the method of Entschel and Karrer s4 as in the technical synthesis of canthaxanthin (see ref. 158). Alternatively, the oxygen functions can be present in the end-groups before the C40-skeleton has been entirely built up, as in the synthesis of canthaxanthin by Warren and Weedon. a32 190

Recent Progress in CarotenoidChemistry MgBrj

~

HO

C

~

%c °" I

MgBr GRIGNARD REACTION

P-C19- Aldehyde

C40-Di°l

/t,

ALLYL REARRANGEMENT AND DEHYDRATION

15,15'- Dehydroi~_ c arotene

/ t

PARTIAL REDUCTION CI5-TRAN$ ISOMERIZATION

~3-Carolene

FIG. 12. The technical synthesisof/3-carotene.149 Non-allylic hydroxyl groups in 3-positions, a very common arrangement in naturally occurring xanthophylls, have generally been present in the cyclic end-group before the entire formation of the C40-molecule. Recently, aliphatic carotenoids hydroxylated in 1-positions have been synthesized. For this purpose a convenient synthesis in high yield was reported by Surmatis and Ofner,317 which involved addition of water to a Wittig salt to form the tertiary alcohol:

~

CHzP ( C.,6Hs)3Br

HO~/C

HzP ( CGH5)~B r

H20

This reaction was used in the synthesis of 1,2,1',2'-tetrahydro-l,l'-dihydroxylycopene (XXIII), and later, by Bonnett, Spark and Weedon,z4 for the synthesis of rhodopin (XXII). An alternative method for the synthesis of the phosphonium salt was also used by the latter authors. 24 Spirilloxanthin (XXVIII), methoxylated in the 1-positions, was recently synthesized by Surmatis and Ofner;317 the methoxyl group being introduced at the 191

Progress in the Chemistry of Fats and other Lipids

Table 14. Totally synthesized naturally occurring carotenoids Class

Designation

Structure

References

CAROTENES

Phytoene Phytoftuene ~-Carotene Neurosporene Lycopene

(I) (I1) (111) (IV) (V)

Bisdehydrolycopene

(VII)

Monocyclic

7-Carotene ~-Carotene Torulene Chlorobactene

(IX) (XI) (X) (XII)

Bicyclic

fl-Zeacarotene a-Zeacarotene E-Carotene

(Vlll) (Vllla) (Xlll)

a-Carotene E-Carotene Isorenieratene Renieratene Renierapurpurin

(XIV) (XV) (XVI) (XVll) (XVIII)

Aliphatic

74 74 74 74 182, 144, 266, 277, 40 317 100, 280 141 , 280 24 280, 33 333 132, 178, 136, 135, 149, 133, 142, 155, 266, 316, 279, 154, 251, 138, 277, 40 180, 88, 139, 279 179, 324 344,199, 53 345, 53 345, 53

HYDROXY AND METHOXY CAROTENOIDS

Aliphatic

Chloroxanthin (lXX) Rhodopin (XXII) 1,2,1 ',2'-Tetrahydro-1,1'-! dihydroxy-lycopene ! (XXIII) (XXVlll) Spirilloxamhin

333 24

Monocyclic

OH-Chlorobactene

(XXXlll)

24

Bicyclic

Cryptoxanthin

3-hydroxy-flcarotene 3,3'-dihydroxy-flcarotene zeaxanthin dipalmitate

147

Zeaxanthin Physalien

317 317

147,148,357,155 148, 357, 155

CARBONYL-CAROTENOIDS

Aliphatic

Apo-8'-lycopenal (Ca0) Bixin (as methylester) Crocetin (as methylester)

192

(XXXW)

24 1, 137, 145, 266, 34 134, 34, 145, 266

Recent Progress in Carotenoid Chemistry

Table 14. Totally synthesized naturally occurring carotenoids--contd. Class Monocyclic

Designation

Structure

fl-Apo-10'-carotenal (C27) (XL) /3-Apo-8'-carotenal (Ca0) (XLI) (XLIII) Y,4'-Dehydro-17'-oxo~,-carotene Torularhodin (XLIV)

Bicyclic

References

278 278 143 143

3, 332 (XLV) 332, 358, 3 (XLVI) 3,3'-4,4'-tetraketo- 74 /3-carotene

Echinenone Canthaxanthin Astacene

Cs-level by treatment of the methyl-heptenone with sulphuric acid and methylalcohol: CH30H

For the further steps a Reformatsky synthesis, N-bromosuccinimide treatment, lithium aluminium hydride-reduction and finally a Wittig condensation were employed.

3. Totally synthetic carotenoids In the previous section reference has been made to the principal reactions employed for the synthesis of the C40-carotenoid skeleton, and to the mode of introducing oxygen functions in desired positions in the C40-molecule. By these methods about thirty-five naturally occurring carotenoids have been totally synthesized during the last fourteen years. A list of these carotenoids and the major references to the synthetic work has been compiled in Table 14. Naturally occurring carotenoids containing less than forty carbon atoms in the molecule have been included. A number of such carotenoids have also been synthesized, e.g. methylbixin, dimethylcrocetin (diesters of C24 and C20 dicarboxylic acids respectively), as well as a number of vinylogous apo-fl-carotenals and their acids and esters. The latter designation was introduced by Rtiegg, Montavon, Ryser, Saucy, Schwieter and Isler278 for carotenoids with less than forty carbon atoms. The fl-prefix denotes the presence of a/3-ionone end-group. The total number of carbon atoms is usually given in parenthesis, e.g. (C37). Finally, several short-chain carotenoid derivatives, together with carotenoid model substances with more than forty carbon atoms in the molecule have also been synthesized. 193

Progress in the Chemistry of Fats and other Lipids

The latter type of isoprenologue, for instance the unstable decapreno-/3carotene (C60) with nineteen conjugated double bonds synthesized by Karrer and Eugster~81 and later by Isler, Montavon, Rfiegg and Zeller, a54 is not found in nature.

D e c a p r e n o - .8 - c o r o t e n e

Various /3-carotene derivatives with alkyl substituents in the/3-ionone ring have been prepared by Surmatis, Maricq and Ofner.31~ The research group of Hoffmann-La Roche has lately aimed at synthesizing isotope-labelled carotenoids of importance for metabolic studies. This aspect has been reviewed by Isler and Schudel) 58 In this connection it may also be mentioned that Strain, Thomas, Crespi and Katz313 have recently isolated fully deuterated a-carotene, /3-carotene and lutein, obtained from algae grown in heavy water. At present, about seventy synthetic carotenoids have been prepared, often by varying methods. This work has allowed the unequivocal establishment of the chemical structure of a great many carotenoids, and, by comparison with synthetic material, has enabled the identification of trace carotenoids. The achievements in the synthetic field form the foundation for the technical synthesis of/~-carotene, used as a provitamin A and for foodstuff colouring, and for the other technically important carotenoids (canthaxanthin, /3-apo-8'carotenoic ester and aldehyde (C30) and torularhodin methyl ester) used as food colours.

B. Partial synthesis 1. Introductory remarks It has been possible by means of particular reactions to carry out partial synthesis of a number of carotenoids. Reference to such reactions will be made below.

2. Double bond introduction Dehydrogenations resulting in double bond introduction (a) between two previously existing double bonds or (b) at the side of the conjugated polyene chain, are efficiently carried out with N-bromosuccinimide, usually in CC14 solution, according to the method first used by Karrer and Rutschmann193 for the formation of bisdehydro-lycopene (VII) from lycopene (V). Further examples of type (a) reaction are the dehydrogenation of the colourless carotenoids phytoene (I), phytofluene (II), ~-carotene (III) and neurosporene (IV), carried 194

Recent Progress in Carotenoid Chemistry out by Zechmeister and Koe. 354 Type (b) examples are found in the dehydrogenation of B-carotene by Zechmeister and Wallcavea56 and by Karmakar and Zechmeister. 176 This field has been thoroughly surveyed by Zechmeister. 35j Especially interesting is the synthesis of bisdehydro-canthaxanthin with two triple bonds presumably in 7,8 and 7',8'-positions, carried out by N-bromosuccinimide treatment of canthaxanthin (XLVI) by Faigle and KarrerP 4 With other solvents the N-bromosuccinimide reaction can be used for the introduction of oxygen functions in the molecule (see below). Dehydrogenations of type (b) have also been performed via BFa-complexes by Bush and Zechmeister. a5 Bodea, Nicoara and Salontai 21 obtained retrodehydrocarotene by action of lead tetraacetate on B-carotene (XIII). Dehydrations of carotenoids containing allylic hydroxyl or alkoxyl groups, by treatment with the acid chloroform reagent as first described by Karrer and Leumann, 192 have been extensively employed for the introduction of conjugated double bonds. This somewhat exceptional reaction has been discussed in Section II. Treatment with phosphoxychloride in pyridine for dehydration of carotenoids was introduced by Surmatis and Ofner, a17 and has lately been used with much success for the dehydration of carotenoids containing the (CHs)2_C(OH)_CH2_CH~_CH2_C(CHs).end.groups.237,164, 234 Rather unexpectedly, Fredriksen 97 observed that the dehydration product of carotenoids with hydroxylated end-groups of the type present in 3,4-dehydro-rhodopin (XXIV) did not exhibit prolonged chromophores.

3. Introduction of oxygen functions Epoxidation of double bonds. Experiments producing 5,6-epoxides from the corresponding carotenoids with B-ionone rings were first conducted by Karrer and Jucker 185 using monoperphthalic acid. Karrer 177 reviewed this field in 1948. Bodea, Nicoara and Salontai 21 obtained mutatochrome (fl-carotenemonofuranoid oxide) on treatment of B-carotene with lead tetraacetate. However, in this experiment a 5,6-epoxide was not considered as an intermediate. Introduction of hydroxyl and ester groups. By performing the N-bromosuccinimide reaction in the presence of glacial acetic acid, Entschel and Karrer s4 demonstrated the formation of 4,4'-diacetoxy-B-carotene from B-carotene (XIII). Karrer and Jaeger 183 synthesized 3,4'-dihydroxy-B-carotene from cryptoxanthin (3-hydroxy-B-carotene) by the same method, but attempts to synthesize 3,3',4,4'-tetra-hydroxy-B-carotene from zeaxanthin (3,3'-dihydroxy-Bcarotene) have so far been unsuccessful. By hydrolytic cleavage of BF3-complexes, various hydroxylated carotenoids have been synthesized from hydrocarbons. Wallcave and Zechmeister a3° obtained isocryptoxanthin (4-hydroxy-B-carotene) from retrodehydro-B-carotene by BF3-treatment. In a similar manner Bush and Zechmeistev~5 produced 5,6,5',6'-tetra-hydroxy-lycopene from lycopene (V) and 4-hydroxy-7-carotene, in low yield, from ~,-carotene (IX). Bodea, Nicoara and Salontai 21 have reported 195

Progress in the Chemistry of Fats and olher Lipids

the formation of isocryptoxanthin and isozeaxanthin by action of lead tetraacetate on /3-carotene. Esterification of secondary hydroxyl groups is readily obtained at room temperature with the acid chloride or anhydride in pyridine solution (see, for example Kuhn and Sorensen 211 and Zechmeister and Cholnoky. 353 Carotenoids with tertiary hydroxyl groups are only acetylated in about l0 per cent yield under these conditions. 92v Introduction of ether groups. According to Entschel and Karrer s4 allylic ether groups can be introduced by treatment with N-bromosuccinimide in ethanol-containing CHC13. Bush and Zechmeister 35 obtained 4-ethoxy-acarotene by ethanolysis of a-carotene-BFz, and Wallcave and Zechmeister 33° demonstrated the formation of 4-methoxy-/3-carotene and 4,4'-dimethoxy-/3carotene on methanolysis of retrodehydrocarotene-BF3. According to Wallcave and Zechmeister, aa0 carotenoids containing allylic hydroxyl groups can undergo smooth formation of the corresponding methyl ether in acid methanol. Methylation of non-allylic hydroxyl groups can be carried out according to the conventional procedure by Karrer and Takahashi tgs or better according to the method of Kuhn, Trischmann and LSw '~12as demonstrated by Liaaen-Jensen 227. Introduction of keto groups. Petracek and Zechmeister 2as obtained echinenone (XLV) and canthaxanthin (4,4'-diketo-/3-carotene) by N-bromosuccinimide treatment of /?-carotene (XII1) in ethanol-containing CHCl3-solution. After hydrolysis of the BF3-complex of a-carotene, Bush and Zechmeister a5 isolated 4-keto-a-carotene. Entschel and Karrer s6 obtained rhodoxanthin (3,3'-diketodehydro-retro-fi-carotene) on manganese dioxide oxidation of zeaxanthin. Retrodehydro-zeaxanthin is assumed to be an unstable intermediate in this reaction. Davis and Weedon 75 have described an elegant synthesis of astacin (3,4,3',4'-tetraketo-/?-carotene) from canthaxanthin (4,4'-diketo-/?-carotene) by air oxidation in strong alkaline conditions. The various methods for oxidation of hydroxyl groups to keto grcups have been dealt with in Section II. V. BIOCHEMICAL ASPECTS

A. Function Detailed discussions of the function of carotenoids have been given by Goodwin,102,104, 109, 110 Stanier299, 300 and Mackinney. 248 Carotenoids are widely distributed in the vegetable and animal kingdoms. A rather striking feature is that all photosynthetic organisms synthesize carotenoids, and that these pigments are located in the photosynthetic units in close association with the chlorophylls. That carotenoids in many cases participate in photosynthesis by absorbing light energy which subsequently is transferred to chlorophyll has been demonstrated by Dutton, Manning and Duggar 79 and Duysens. so The role of carotenoid pigments in phototaxis is well documented, and has been thoroughly reviewed by Clayton 49 and Goodwin. 109 The function of carotenoids as protectors against photodynamic destruction was revealed by the work of Griffiths, Sistrom, Cohen-Bazire and Stanier, 117 196

Recent Progress in Carotenoid Chemistry based on studies carried out with mutants of a photosynthetic bacterium devoid o f coloured carotenoids. Such a killing effect was also observed by Cohen-Bazire and Stanier 52 on exposure of bleached cells of Rhodospirillum rubrum to light and air. These cells lacked coloured carotenoids owing to growth in the presence o f diphenylamine. According to the work of Kunisawa and Stanier, 215 Mathews and Sistrom, 250 and Dundas and Larsen, 7s a similar function as protectors against excessive irradiation can be attributed to carotenoids in pigmented, nonphotosynthetic bacteria. In mammalian physiology, provitamin A activity is a well-established function of coloured carotenoids containing at least one unsubstituted fl-ionone ring. Islet and Zelled 59 have published an interesting discussion of the relationship between vitamin A activity and the chemical structure of the carotenoids. Introduction of an extra double bond in 3,4-position of the fl-ionone ring, an additional methyl group in 2-position, substitution of one of the gem-methyl groups with an ethyl group, epoxidation in 5,6-position, or introduction of cis double bonds in the polyene chain, reduce the vitamin A activity, but do not completely destroy the biological activity. Other functions of the carotenoids have been claimed, but are not definitely proved. These include possible participation in oxygen transfer, as discussed by Calvin, 36 Cholnoky, Gybrgyfy, Nagy and P~ncz61, 4z Krinsky, 202 Yamamoto, Nakayama, Chichester 34s and others; for references see Yamamoto, Chichester and Nakayama. a46

B. Biosynthesis Various aspects of this topic have been surveyed in many recent reviews, for example Goodwin, TM, 109, m b Stanier,300 Mackinney,24s Porter and Anderson 26s and Liaaen-Jensen. 232 The problem may be conveniently subdivided as follows: (1) The nature of the primary Cs-precursor (2) The biosynthesis of the colourless C4o-unit from the Cs-precursor (3) Dehydrogenation of the C40-unit to coloured carotenoids (4) The cyclization step (5) The aromatization reaction (6) The introduction of oxygen functions: (a) epoxy groups; (b) hydroxy, ether and ester groups; (c) keto functions (7) The biosynthesis of apo-carotenoids. One knows at present the broad outline of the path of carotenoid biosynthesis, although a number of points need further documentation. The available evidence indicates that a common biosynthetic pathway is valid for all carotenogenic systems, and that only variations in the ultimate steps (4)-(6), lead to the carotenoids characteristic of various types of organisms.

1. The nature of the primary Cs-unit Like other natural products with plenary isoprenoid skeletons, such as 197

Progress in the Chemistryof Fats and other Lipids steroids, terpenes and plant rubber, carotenoids are formed from isopentenylpyrophosphate ("active isoprene"). Lycopene formation in tomato homogenates from isotopically labelled isopentenylpyrophosphate has been demonstrated by Varma and Chichester. a29 Isotopic incorporation of mevalonic acid into carotenoids in various systems has been reported by several workers such as Grab, 11s Purcell, Thompson and Banner, 271 Shneour and Zabin, z91 Chichester, Yokoyama, Nakayama, Lukton and Mackinney, 4~ Yokoyama, Chichester and Mackinney, 3~° Braithwaite and Goodwin, 3°, al and Steele and Gurin. 3°1 Although not established in detail for a carotenogenic system, the biosynthesis of the primary Cs-unit appears to proceed as outlined by Lynen, Agranoff. Eggerer, Henning and Mbsleinz46 for squalene biosynthesis (Fig. 13). ~3- Methyl - c rotonyf - C oA

Isovaferyl-

~

CoA

~

Leucin

C02 ATP~ADP

• P

13-Met b y [ - 9 1 u t a c o n y l

-CoA

ATP

2 TPNH -Hydroxy -[~-

rne~ h y l Mevalonic

glutaryl- CoA

-

~

MevaIonic

ADp

2TPN

-5-P

acid

_ ~

ATP

ADP

/2

Acetoace~yl-

acid

Mevalonic

acid - 5

--PP

AT P

CoA

ADP+ P+ C 0 2 ~ / [$opentenyl

Acetyl - CoA

T

Carbohydrates,

_

pp

fa~s

FIG. 13. Pathway for isopentenylpyrophosphate biosynthesis.'247

2. The biosynthesis of the colourless C40-unit from the Cs-precursor By analogy with terpene and steroid biosynthesis, one would predict the biosynthetic transformations of isopentyl-pyrophosphate to proceed via geranyl-pyrophosphate (Clo), farnesyl-pyrophosphate (C15), geranyl-geranylpyrophosphate (C2o) to lycopersene or phytoene (C40), as depicted in Fig. 13 (see Lynch and HenningZ47). In support of this scheme, Anderson and Porter s observed that a 14C-labelled C20-terpenol pyrophosphate (presumably geranylgeranyl-pyrophosphate), synthesized by a soluble, rat-liver enzyme, served as a substrate for phytoene formation in a cell-free tomato preparation. According to Yamamoto, Yokoyama, Simpson, Nakayama and Chicheste~, ~49 farnesylpyrophosphate is also incorporated into carotenoids. 198

Recent Progress in Carotenoid Chemistry i ,r" 0 a.

0

U

~

~<> : < " Z ~ >

" "

e. 0

0

<<

: : il

.~

:

)

I

=:>' ill

I

<>\

i

Z

O ¢-

0 t~ 0

r~

u

\ 0

0

l

0

u~ 0

u~.o

>,

bJ

~c

)

t~

III

0 (U

l

°

0

U

3C U r

"~

- . > -.K o-o

%

o/",o--~ ' 7 11 t ~ j

.o

1

uo>~o ~ u__.O

199

Progress in the Chemistry of Fats and other Lipids There is some controversy about the primary C40-compound: Grob and Boschetti 12° detected lycopersene in Neurospora crassa, and Grob 119 favours the view that lycopersene is a true intermediate in carotenoid biosynthesis, whereas the research groups of Goodwin and Porter have not been able to isolate lycopersene in carotenogenic systems and claim phytoene to be the primary C40product. 72,8 Unequivocal demonstration of the individual step-reactions indicated in Fig. 14 in one-enzyme systems, with their accompanying co-factors, and elucidation of the (Coo + C20) reaction has not yet been carried out.

3. Dehydrogenation of the C4o-unit to coloured carotenoids The stepwise dehydrogenation of phytoene (I) via phytofluene ( I I ) ~-carotene (Ill) and neurosporene (IV) to lycopene (V) was in principle suggested by Porter and Lincoln 269 for lycopene formation in tomatoes, and derived firm support from the kinetic studies reported by kiaaen-Jensen, Cohen-Bazire, N a k a y a m a and Stanier?-35 They recorded the endogenous carotenoid synthesis in cells of Rhodospirillum rubrum, grown in the presence of diphenylamine after removal of the inhibitor. The stepwise decrease in total radioactivity along the biosynthetic route (see Table 15), observed by Anderson and Porter, s when a labelled precursor was used as substrate for carotene formation in tomato plastids, was also consistent with this theory.

Table 15. The synthesis of coloured carotenes .from colourless C4o-precursors (Porter-Lincoln series) Number of double bonds Intermediate

Structure Conjugated

Isolated

Total

Phytoene $ Phytofluene

(1)

3

6

9

Of)

5

5

10

~-Carotene

(Ill)

7

4

11

(IV)

9

3

12

(V)

11

2

13

$ Neurosporene Lycopene

The sequential desaturation occurs by introduction of a new double bond in such a manner that the chromophore is extended by two double bonds at each step. The molecular environment of the reaction site is identical in each of these four dehydrogenation reactions. Discussions, based on mutant studies, have been published by Liaaen-Jensen, Cohen-Bazire and Stanier 238 and by Crounse, Feldman and Clayton 55 on the number of enzymes involved in these transformations. The enzymatic conversion of phytoene to phytofluene by tomato plastids has recently been reported by Beeler and Porter. 17 The possible existence of 200

Recent Progress in CarotenoidChemistry hydroxylated intermediates in these oxidation reactions has been discussed, but has hitherto derived no experimental support. 235, 227,276 4. The cyclization step In contrast to steroids and terpenes, no, or very limited cyclization to cyclohexylidene end-groups takes place in carotenoid biosynthesis. The formation of dihydrofurano heterocycles and cyclopentanone rings is considered to be the result of secondary rearrangement via 5,6-epoxides. The general view is that cyclic carotenoids with fl- and a-ionone rings are formed by cyclization of a C40-compound, but so far no definite knowledge is available about the stage in the biosynthetic chain at which cyclization normally occurs, or about the nature of the cyclization reaction. If phytoene (I) proves to be the primary C40-unit, the limited cyclization could be explained as a result of the rigid, linear shape of the central part of the phytoene molecule. In the original Porter-Lincoln hypothesis269 the biosynthetic route from lycopene (V) to /3-carotene (XIII) was postulated. Decker and Uehleke 76 obtained some conversion of labelled lycopene to /3-carotene by green-leaf plastids. Porter 267 has since demonstrated a similar reaction in particular tomato plastids. Evidence for the conversion of CX4-1abelled phytoene (I) to f-carotene (XI) in a cell-free extract of Staphylococcus aureus has been presented by Suzue. 818 Rabourn 27~ has advanced arguments, based on considerations of molecular models, that E-carotene is the most likely, immediate precursor of the cyclic carotenes. The existence of the monocyclic fl-zeacarotene (VIII) clearly shows that cyclization can occur at other stages than that of lycopene. The failure to detect more saturated cyclic carotenoids may very well be a result of unstatisfactory technique and not of the absence of such compounds. Whether the a-ionone ring is obtained by double bond isomerization of a fi-ionone ring, or by an independent cyclization reaction, is an unsolved problem. 5. The aromatization reaction It seems feasible that carotenoids containing 1,2,3- or 1,2,5-trimethylphenyl end-groups have arisen from the usual methylated cyclohexylidene end-groups by further dehydrogenation accompanied by Wagner rearrangements)3,237 However, supporting biosynthetic evidence is so far lacking. 6. The introduction of oxygen functions The available evidence supports the idea that oxygen functions are incorporated into the carotenoid molecule after the formation of the C40-skeleton. The introduction ofepoxide groups. The 5,6-epoxides are considered as having arisen by oxidation of the corresponding double bonds in fl-ionone rings; see Cholnoky, Gybrgyfy, Nagy and P~ncz61.4z There are several examples of the reverse reaction, which will be dealt with in Section C. Yamamoto, Chichester and Nakayama347 claimed, from tracer experiments with Chlorella vulgaris, 201

Progress in the Chemistry of Fats and other Lipids

that the epoxidic oxygen of violaxanthin is derived from water and not from molecular oxygen. On the other hand, epoxide formation in green plants requires oxygen. This topic has been discussed by the same authors. 34s Plant acids may in certain cases be responsible for the rearrangement of 5,6-epoxides to furanoid oxides. The introduction of hydroxyl, methoxyl and ester groups. The oxygen-dependent introduction of secondary hydroxyl groups in 3,3 '-positions to form xanthophyl Is from a-carotene and fl-carotene has been nicely demonstrated by Claes. 5° Tertiary hydroxyl groups in 1-position as present in many bacterial carotenoids seem to be the result of the hydration of a double bond. z27, 236 The latter reaction occurs in the presence of oxygen as well as under presumably anaerobic conditions. Methoxyl groups in the above positions are apparently introduced by subsequent transmethylation of the tertiary hydroxyl groups. TM z27, ,_,36 Braithwaite and Goodwin 29 demonstrated that formate served as a carbon source for the methoxyl groups in spirilloxanthin (XXVIII), synthesized in

Rhodospirillum rubrum. It is a plausible supposition that ester groups have arisen by esterification of (i) carotenoid alcohols or (ii) acids. Of type (i), esterification, esters of long-chain fatty acids are commonly encountered. However, fucoxanthin is an acetate. 171 Of type (ii) methyl-esters only are known. The introduction of keto groups. The formation of conjugated keto-groups in carotenoids of the spheroidenone (XXVI) type is, according to the investigations of van Niel, z26 Cohen-Bazire, Sistrom and Stanier, 51 Shnoeur, 289, ~90 Eimhjellen and Liaaen-Jensen, s2 a strictly oxygen-dependent, enzymatic reaction, where no hydroxylated intermediates have been detected.

Conjugated keto groups in carotenoids synthesized by obligatory anaerobic bacteria must be introduced by a different mechanism. 2a2 In the capsanthin (XLVII)-type carotenoids the introduction of the keto group is considered as the result of a pinakolin rearrangement of the hypothetic diol intermediate, as has been discussed by Entschel and Karrer. s6

OH

Thommen and Wackernagel a21 assumed fl-carotene (XIII) to be the biosynthetic precursor of echinenone (XLV) and canthaxanthin (XLVI) in Crustaceae. The formation of rhodoxanthin (3,3'-diketo-retro-fi-carotene) from lutein 202

Recent Progress in CarotenoidChemistry (3,3'-dihydroxy-a-carotene) in Reseda odorata has been observed by Stokke; see Sarensen. a19 The plausible suggestion that the carboxyl group of torularhodin (XLIV) is introduced by oxidation of a methyl group of torulene (X) has been put forward by Winterstein.336

7. The biosynthesis of apo-earotenoids It is generally believed that apo-carotenoids (carotenoid pigments containing less than forty carbon-atoms in the carbon skeleton) have arisen by oxidation of C40-carotenoids. This assumption is in accordance with the fact that such apo-carotenoids invariably have carbonyl-functions in one or both ends (aldehydes, acids or esters). The apo-carotenoids can therefore be considered as metabolic products of the C40-carotenoids, and will be discussed below. C. Metabolism Surprisingly little is known about the metabolic fate of carotenoids. Glover and Redfearn1°1 and Fazakerley and Glover95 have claimed that/3-carotene is transformed into vitamin A in the mucosa of the intestinal tract by oxidative degradation starting from one end of the molecule, and not, as previously assumed, by oxidative attack of the central double bond of/3-carotene. The demonstration by Winterstein, Studer and Rfiegga~8 of the natural occurrence of various/3-apo-carotenals lends support to such a hypothesis: /3-carotene (C40) (XIII) /3-apo-8'-carotenal (Ca0) (XLI) /3-apo-10'-carotenal (C27) (XL) /3-apo-12'-carotenal (C25) Vitamin A aldehyde (Cz0) Vitamin A (Cz0) Similarly, various apo-lycopenals can be considered as metabolic oxidation products of lycopene. However, available evidence has been further authoritatively discussed by Glover 101b and Olson mb and the general conclusion was reached that the above-mentioned scheme does not represent the main conversion process of/3-carotene to vitamin A. In vivo transformations of carotenoid epoxides to the corresponding carotenoids containing double bonds in 5,6- or 4,5-positions have been demonstrated by many workers. Liaaen-Jensen and Sorensen219 reported the transformation of violaxanthin to zeaxanthin in brown algae, and Krinskyz0z demonstrated the conversion of antheraxanthin to zeaxanthin in Euglena graeilis. Yamamoto, Nakayama and Chichesteraaa observed similar reactions on illumination of 203

Progress in the Chemistry of Fats and other Lipids

g

/

o

o ~2

/, 'E a ~6

204

o I

.r o

Kecent Progress in Carotenoid Chemistry spinach a n d lima beans. Sapozhnikov, Maevskaya, K r a s o v s k a y a - A n t r o p o v a , Prialgavskaite a n d T u r c h i n a28a have shown that v i o l a x a n t h i n a n d lutein in leaf are in equilibrium. Observations by Blass, A n d e r s o n a n d CalvinIs supported the interrelationship o f the two latter pigments in algae.

VI. REFERENCES

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