The Carotenoid Group

Chapter 7

The Carotenoid Group j. 13. DAVIS

The carotenoids are an important class of natural fat-soluble colouring matters usually yellow or red in colour which are widely distributed in both the animal and vegetable kingdoms. They are polyisoprenoid (often C4o) and are characterised by a chromophore of numerous (frequently ten or eleven) conjugated double bonds. In many members of the class the conjugated chain is terminated at one or both ends by a carbocyclic ring, for which reason they are conveniently discussed with alicyclic compounds. Some carotenoids possess the important property of acting as precursors of vitamin A when ingested by the animal body. In recent years many of the carotenoids have been synthesised. Several carotenoids, including//-carotene and//-apo8'-carotenal, are manufactured on an industrial scale for use as food-colouring matters, those mentioned here being, in addition, potent provitamins A. A suspension of microcrystalline//-carotene in edible fat is incorporated into margarine to give it the required vitamin A potency; /J-carotene although water-insolublecan be used to colour aqueous media if dispersed in a very fine emulsion (cf. Isler and co-workers, Chimia, 1961, 15, 208; R. H. Bunnell et al., Food Technol., 1958, i2, 527, 536; 1962, i6, 76; Adv. Food Res., 1966, x5, 195). 1. Basic constitution, classification and nomenclature

(a) Basic structure and constitution Each of the C40 carotenoids consists of a central C22 branched chain to each end of which is attached a C9 end-group; in any one carotenoid the two end-groups may be either the same as in//-carotene (I) and in lycopene (III), or different as in x-carotene (II). In most carotenoids the central C22 chain is a fully unsaturated nonaene unit (as in formulae I to v) but occasionally it contains two extra hydrogen atoms (as for example in neurosporene,

232

THE CAROTENOID GROUP

7

p. 27 6) and occasionally two (or four) fewer--as in the four carotenoids described by A . K . M a l l a m s et al. (Chem. Comm., 1967, 3oi), which have acetylenic bonds at C(~)-C(8) and/or at C(~,)-C(v). EThe naturally-occurring furanoid oxides (p. 293), which appear to have a modified central C~2 unit, are in vivo rearrangement products of the corresponding epoxides, which have normal C22 units]. Little variation occurs in the central part of the carotenoid molecule, but the end-groups show considerable diversity. tt

i

/9-Carotene

(I)

a-Carotene (/T)

l..u

CI~)

Thus they can be either acyclic [e.g., as in lycopene (III)] or cyclic, the most common ring systems being the /~-ionone type Eas in /~-carotene (I) ~ and ~-ionone type (cf. II). In addition, each of the above types of end-group can carry one or more of a variety of oxygen functions, e.g.

~ o

o

Types of end-group of less common occurrence are those containing a benzene ring, a cyclopentane ring, and a cyclohexenylidene ring, these being * Carotenoids with acyclic end-groups are often written as in formula III to emphasise the structural relationship they bear to those carotenoids with cyclic end-groups; however they are more accurately represented by the linear form (as in formula XI; p. 2 3 4 ) .

I

BASIC STRUCTURE

233

AND CONSTITUTION

exemplified by isorenieratene (IV), capsorubin (V), and rhodoxanthin (vI), respectively.

]sorenieratene (ISD OH

L

Capsorubin 0E) OH O

O

Rhodoxanthin 03I)

As yet only two carotenoids (rhodoxanthin and the corresponding diol, eschscholtzxanthin) with the cyclohexenylidene (or "retro"*) type of endgroup have been definitely identified in nature" such --- molecules have a modified central C22 unit due to the introduction of further unsaturation (as have also the A ~ end-group allenic carotenoids, p. 328). Finally, several polyenes have been isolated from the vegetable kingdom which contain fewer than 4o carbon atoms but which bear an obvious structural relationship to the C40 carotenoids and are usually included with them. These "apocarotenoids" probably arise by in vivo oxidative degradation of C40 carotenoids and carry aldehyde or carboxyl groups" examples are/5-apo-8'-carotenal (C30, vii), azafrin (C27, viii), and crocetin (C20, IX). Similarly, vitamin A (C20, X) is included as it is produced in the animal body from ingested carotenoids, and still contains one half of the/5-carotene carbon skeleton:

V

~

B- Apo-8'- carotena[ (3ZID

CHO

* Termed "retr o" by I47. Oroshnik e! al. (J. Amer. chem. Soc., 19.52, 74, 295) to signify t h a t the double bond s y s t e m had m o v e d b a c k one position into the ring end-group.

234

THE

~

CAROTENOID

7

GROUP

CO2H Azafrin (~:711T)

H 0 2 C ~ / C 0 2 H Cr'ocetin

~ C H 2 O H VitaminA (~) The constitution of every carotenoid so far discoveIed is in accordance with L. Ruzicka's biogenetic isoprene rule (cf. Proc. chem. Soc., 1959, 353)Thus lycopene contains eight isoprene units arranged in two groups of four, the isoprene units being joined head to tail within each group of four whilst the two groups are joined tail to tail: i

I

Lycopene (ZI) (-lID

and in a structural sense all other carotenoids can be derived from this molecule [by cyclisation of the end-groups (cf. III), insertion of oxygen functions (cf. p. 232), double-bond migrations, etc.]. Whereas most of the carotenoids obey the classical isoprene rule, the special premises of the biogenetic isoprene rule (involving alkyl shifts) have to be invoked in the case of the trimethylphenyl- and trimethylcyclopentyl- end-groups (cf. P-345). (b) Classification and nomenclature

The carotenoids can be classified into four structural types: the hydrocarbons (or "carotenes"); those carrying hydroxyl, carbonyl, methoxyl, or epoxide functions; the xanthophyll esters (natural esters, e.g., palmitates, of hydroxylic carotenoids) ; and the carotenoid acids.The term "xanthophyll" is used as a generic name to denote, nowadays, any naturally-occurring carotenoid containing an oxygen function (originally the term was restricted

I

235

C L A S S I F I C A T I O N AND N O M E N C L A T U R E

to those carotenoids containing a flee hydroxyl group: R. Kuhn et al., Z. physiol. Chem., 1931, 197, 141). In addition P. Karrer has used the term xanthophyll for a specific carotenoid XII, now usually called lutein: ,OH

HO

Lutein (= Karrer,s 'xanthophyll')

Carotenoids can also be roughly classified according to their behaviour on partition between the two immiscible solvents petrol and 90 % (occasionally 95 %)methanol. The hydrocarbons, the xanthophyll esters, and carotenoids carrying only ether functions or a single keto group appear in the upper (petrol) layer, or epiphase (epiphasic carotenoids). Carotenoids carrying two or more hydroxyl groups appear in the lower layer, or hypophase (hypophasic carotenoids). Monohydroxy- and diketo-carotenoids and the carotenoid acids appear in both layers. (The exact distribution of a carotenoid between the two layers can be determined spectrophotometrically and the epiphase: hypophase ratio then used as an aid to the elucidation of structure: see p. 245.) Carotenoid formulae are n u m b e r e d according to a scheme first proposed by K a r r e r and since t h e n a d o p t e d and developed by the I.U.P.A.C. Commission. The carbon a t o m s carrying the geminal m e t h y l groups are given the numbers I and I ' and each half of the carbon chain (of a C,0 carotenoid) is n u m b e r e d i - x 5 and 1'-15'. In an u n s y m m e t r i c a l carotenoid, the plain numerals are reserved for t h a t half of the molecule containing a fl-ionone t y p e of end-group. Thus 7-carotene is n u m b e r e d : 18' 16

17

19

Me Me

~

Me

20

Me

Me

4'

~"",3'

"Me

In a carotenoid having one end-group of the a-ionone t y p e and the other acyclic, the carbon a t o m s in t h a t half of the molecule which includes the ring are given plain numerals. Cyclopentane-type end groups are n u m b e r e d :

OH

236

THE CAROTENOID

GROUP

7

If necessary, the methyl substituents are numbered as above. The apocarotenoids are now numbered as illustrated below: 0

,~ -Apo- 8'- car'otenat

(In papers and reviews on carotenoids which appeared up to about 195o a different system of naming apocarotenoids was used. In this, the figure following the prefix "apo" indicated the number of double bonds which had been removed - usually by oxidative degradation- from the original molecule, so that using this system the compound above would have been called fl-apo-2-carotenal).

(c) Conformation Light-absorption (see p. 253; also, E. A. Braude et al., J. chem. Soc., 1952, 1419) and P.M.R. (B. C. L. Weedon et al., ibid., 196o, 2870 ) data indicate that in carotenoids containing a fl-ionone type of end-group the ring double bond (and hence the ring itself) is not coplanar with the polyene chain but lies at an angle to it .Whether the preferred conformation of carotenoids of this type approximates more closely to the s-cis or to the s-tram form ("cis" or "tram" about the 6,7 single bond) is unknown:

s - trans

s- cis

X-ray investigations of all-trans-IS,I5'-dehydro-fl-carotene (cf. formula I) and of one of the crystalline forms of all-tram- vitamin A acid (cf. p. 286) have shown that in the crystalline state both of these compounds exist in conformations which approximate more close]y to the s-cis form (W. G. Sly, Acta Cryst., 1955, 8, 115; C. H. Starn and C. H. MacGillavry, ibid., 1963, 16, 62). Formulae are written here in the s-cis form, as in the majority of original papers so far published. 2. Occurrence, function

and

isolation

(a) Occurrence The carotenoids are one of the most widespread groups of naturallyoccurring colouring matters, being found in plants, algae, fungi, bacteria and

2

OCCURRENCE

237

in many forms of animal life. Many of the yellow, orange, and red colours observed in the vegetable and animal kingdoms are due to them, familiar examples beil~g the yellow of egg yolk, the red of the tomato, the orange colour of the common marigold and the yellow of the buttercup, the pink colour of the flamingo and the yellow of the canary. In addition, carotenoids are present in green leaves, grass, and seaweed where their presence is masked by chlorophyll. Carotenoids are basically plant colouring matters in the sense that animals are unable to synthesise them de novo from "acetate" (as do plants and bacteria) but derive them from their food (which ultimately is of vegetable origin). Animals frequently modify ingested carotenoids (e.g. by inserting oxygen functions into the end-groups, or in certain cases by degrading them to vitamin A). So far about 18o carotenoids have been individually recognised and the structures of about half of these are known with a considerable degree of certainty. In addition many more colouring matters have been isolated in very small amounts often as the minor components of a complex mixture of carotenoids (but not all of these are necessarily genuine: see p. 241). Most natural sources yield a mixture of several carotenoids with a few of them (the "major carotenoids") markedly predominating over the remainder. The yield of even the major carotenoid(s) generally amounts to only 0.02 to o. 1% of the dry weight of the material extracted although yields of I to 2 % are obtained very occasionally; an exception is the deep red fringe of the corona of the pheasant's-eye narcissus (N. majalis) 16% of the dry weight of which consists of fl-carotene (V. H. Booth, Biochem. J., 1963, 87, 238). In recent years some order has emerged from the many reports dealing with the natural occurrence of carotenoids (cf. T. W. Goodwin, Adv. Enzymol., 1959, 21, 295; "The Comparative Biochemistry of the Carotenoids", Chapman and Hall, London, 1952; H. H. Strain, J. Amer. chem. Soc., 1948, 70, 1672). Thus the green leaves of all plants contain a mixture of carotenoids consisting of 20 to 4o% of carotenes and 60 to 80% of xanthophylls. The carotene fraction consists of fl-carotene accompanied by a small and variable amount (o to 35%) of a-carotene. The fl-carotene content usually falls in the range 20 to 7 ~ mg./IOO g. dry weight. The xanthophyll fraction consists of a mixture of lutein, violaxanthin, and neoxanthin often accompanied by small amounts of kryptoxanthin and zeaxanthin and, exceptionally, taraxanthin (all hydroxylic carotenoids). The other pigments originally reported to occur in leaves (Strain, "Leaf Xanthophylls", Washington, 1938 ) were probably artefacts produced during isolation. The leaves of a plant, which usually represent less than half its total weight, contain nearly all (more than 90%) of the total carotenoid. (Regarding the rate of carotenoid synthesis in growing plants, see Goodwin, loc. cir.). The concen-

238

THE CAROTENOID

GROUP

7

tration of carotenoids in many fruits increases dramatically during ripening. Certain carotenoids or groups of carotenoids are largely restricted in their occurrence to certain classes of plant or animal life. Thus the photosynthetic organisms may be broadly classified according to the nature of their major constituent carotenoids, as follows (cf. Goodwin, los. cit., 1959; J. gen. Microbiol., 1957, 17, 467; Strain, "Chloroplast Pigments and Chromatographic Analysis", 32nd. Ann. Priestley Lectures, Pennsylvania State Univ.~ I958) : Natural source x. Green tissues of higher plants 2. The algal classes Chlorophyceae, Xanlhophyceae, Euglenineae, Rhodophyceae 3. Brown algae (Phaeophyceae) and diatoms (Bacillariophyceae) 4. Blue-green algae (Cyanophyceae) 5. The algal class Dinophyceae 6. Purple bacteria (Thiorhodaceae and Athiorhodaceae)

Major carotenoids /5-Carotene, lutein, neoxanthin, violaxanthin Rather variable: quite often as for green tissues /5-Carotene, fucoxanthin fl-Carotene, echinenone, myxoxanthophyll fl-Carotene, peridinin Lycopene and allied methoxy- and hydroxy-derivatives (p. 306).

In contrast, flower petals and fruit (which are non-photosynthetic) contain a wide variety of carotenoids and some carotenoids have as yet been found in only one species (i.e. are apparently species-specific). Whereas the xanthophylls in leaves occur unesterified those in flowers and fruit usually occur esterified with fatty acids like palmitic acid. Lists of carotenoids isolated from natural sources have been given by P. Karrer and E. Jucker ("Carotenoids", Elsevier, Amsterdam, I95O), Goodwin (1952, op. cit. and 1959, loc. cir.) and, for the vegetable kingdom only, by W. Karrer ("Konstitution und Vorkommen der organischen Pflanzenstoffe", Birkh~user, Basel, I958 ), Strain (loc. cir. ; J. Amer. chem. Soc., 1948, 70, I672), T. O. M. Nakayama (in "The Physiology and Biochemistry of the Algae", Ed. J. Myers, Academic Press, New York, I963) , and M. B. Allen et al. (J. gen. Microbiol., 1964, 34, 259). D. L. Fox has. discussed animal carotenoids (in "Symposia on Comparative Biology", Vol. I, Ed. M. B. Allen, Academic Press, 196o ). Lutein, astaxanthin, rhodoxanthin, zeaxanthin and several carotenoids of unknown structure (including the widespread feather carotenoid canaryxanthophyll) have been isolated from the feathers of various birds (cf. O. V61ker, Fortschr. Chem. org. Naturstoffe, 196o, i8, 177). Several caroten-

2

OCCURRENCE

239

oids (including a- and fl-carotenes, lutein, echinenone, spirilloxanthin, and myxoxanthophyll) have been isolated from both freshwater and marine sediments. Some of these sediments were formed (by the accumulation of carotenoid-containing plant debris) about 20,000 years ago, so demonstrating the comparative stability of some carotenoids in these anaerobic conditions (cf. J. R. Vallentyne in "Symposia on Comparative Biology" Vol. I, Ed. Allen, 196o, p. 96). Many of the carotenoids isolated without the aid of column chromatography, and this includes all those reported prior to 1931, were probably not pure and so only limited significance can be a t t a c h e d to this earlier work. Carotenoids usually occur in n a t u r e in the all-trans configuration although carotenoids containing one or two of their double bonds cis are also known (since some carotenoids undergo stereoisomerisation very readily the isomer isolated m a y not always be the one originally present). Lycopene, y-carotene, and neurosporene sometimes occur in polycis forms (for list of sources, see L. Zechmeister, Fortschr. Chem. org. Naturstoffe, 196o, 18, 282). The carotenoids are not themselves water-soluble but are frequently found in aqueous media where they exist in the form of water-soluble protein complexes ("chromoproteins")*. For this reason carotenoids cannot be ext r a c t e d from homogenised green tissues (e.g. leaves) by non-polar solvents but are readily extracted following denaturation of the protein with a polar solvent (e.g. methanol). The colouring matters revealed in the leaves of deciduous plants in a u t u m n following the destruction of the chlorophyll are frequently derived from the carotenoids present in the green (summer) leaves, their exact nature depending on the extent to which the summer carotenoids are modified during the period when the chlorophyll is destroyed. [This in turn probably varies from one species to another, although there does appear to be a general tendency for the xanthophylls to be esterified and for the fl-carotene to be destroyed (Goodwin, Biochem. J., 1958 , 68, 503; E. C. Grob and W. Eichenberger, ibid., 1962, 85, l i P ) ] .

(b) Function The function of certain carotenoids as vitamin A precursors and their connection with visual processes are mentioned on pp. 283 and 289. The extent to which the carotenoids are involved in other facets of the metabolism of plants and animals is still unknown; various natural functions have been postulated but have rarely received experimental proof (cf. Goodwin, Ann. Rev. Plant Physiol., 1961, 12, 237). Two examples which do have some foundation are: (a) in photo* Little is known of the structures of these complexes as few of them have as yet been obtained pure; cf., however, p. 321.

240

THE

CAROTENOID

GROUP

7

synthesis - carotenoids (either as such or as their protein complexes) absorb light strongly in those regions of the visible spectrum where chlorophyll does not absorb and may be able to transfer the absorbed light energy to the chlorophyll, and (b) as a protective influence- if certain carotenoid-rich bacteria are artificially deprived of their carotenoids and then exposed to air and light they are rapidly killed (G. Cohen-Bazire and R. Y. Stanier, Nature, I958, 181, 250; cf. also L. J. Wright and H. C. Rilling, Photochem. Photobiol., I963, 2, 339; H. Claes et al., Z. lXIaturforsch., I96I, x6b, 445; 1959, x4b, 746; C. Bril, Biochim. Biophys. Acta, 1962, 66, 5o). (c) Isolation of carotenoids from natural sources; chromatography In the isolation of carotenoids from plant material it must be remembered that when excised plant material is stored, a large proportion of any carotenoids therein is gradually destroyed. In green leaves at least two mechanisms operate: enzymatic oxidation, which requires molecular oxygen; and, of less importance, photo-destruction, which is favoured by, but not entirely dependent on, molecular oxygen (R. B. Gri~th and C. R. Thompson, Bot. Gaz., 1949, i i i , 165). Freshly collected material must therefore be used if an accurate survey of the carotenoids present is to be obtained. Any preliminary slicing of the material must be followed by its immediate immersion in the extracting solvent as enzymatic oxidation of the carotenoids is greatly accelerated in plant cells which have been damaged (Booth, Qual. Plant. Mat. Veg., 1958, 3-4, 317). From relatively dry material the carotenoid colouring matters are extracted from it either by blending with methanol, acetone or methanol-petrol, which also dehydrate the material, or by grinding with sodium sulphate and sand and stirring the resultant mixture with ether; carotenoids in juices expressed from fruits can be extracted by adding filter aid, which adsorbs the carotenoids, filtering and extracting the filter cake with acetone; whole fruit can be processed similarly after first blending with aqueous methanol. The crude extracts are then diluted with ether or petrol, sufficient brine is added to give two layers, and the upper (solvent) layer is separated and worked up as follows. Lipids and chlorophyll are removed by (cold) saponification (in, e.g., aqueous ethanol or ethermethanol). The mixture is then diluted with brine and the carotenoids, except any acids present, are extracted with ether or petrol. At this stage the mixture is usually subjected to partition between petrol and 90% methanol (see p. 235), the epiphasic carotenoids and the hypophasic carotenoids subsequently being treated independently. (If the mixture is partitioned prior to the saponification step the epiphase obtained includes the xanthophyll esters. If this epiphase is saponified and the mixture then partitioned, the hypophase contains those xanthophylls which were originally present

2

ISOLATION

FROM

NATURAL

SOURCES

241

as xanthophyll esters). The epiphasic and the hypophasic fractions are then resolved into their individual constituents by column chromatography (cf. below). The quantity of carotenoid present in various fractions obtained during the isolation procedure is readily estimated spectrophotometrically (using the extinction coefficient of a known carotenoid which absorbs in the same spectral region). In certain cases the saponification step is omitted, the chlorophyll being removed by two-stage chromatography (on powdered sucrose followed by magnesia; Strain, op. cit., I958, E. M. Bickoy et al., J. agric. Food Chem., 1954, a, 563). This does not necessarily yield lipid-free samples but is useful when the carotenoids are to be estimated spectrophotometrically (rather than isolated as solids) and when alkali-labile carotenoids (e.g., fucoxanthin, astaxanthin) are to be isolated. It also allows the crude mixture of colouring matters extracted from a natural source to be resolved into its components in a very short time so minimising the formation of artefacts (see below). Exposure to acid must be avoided and exposure to heat, light, and air kept to a minimum throughout any isolation procedure. Many variations of this general scheme are used (cf. Goodwin in "Modern Methods of Plant Analysis", Eds. K. Paech and M. V. Tracey, Springer, Berlin 1955; Strain, op. cir. 1958; A. L. Curl et al., J. agric. Food Chem., 1953, I, 456; J. Food Sci., 1962, 27, 537; 1961, 26, 442). Countercurrent distribution has also been used (Curl and G. F. Bailey, J. agric. Food Chem., 1953, i, 456; and refs. below); it offers a convenient way of making a preliminary separation of a complex mixture into groups of carotenoids with similar polarities. Distribution between petrol and 99 % methanol is first used and resolves the mixture into three fractions--hydrocarbons, mono-ols, and di-ols + polyols; the diol + polyol fraction is then further fractionated using petrol~73.5 % methanol into monoepoxy-diol, diepoxy-diol, and polyo! fractions. Other solvent systems have also been used (Curl et al., ibid., I96o, 8, 356; z962, IO, 504; J. Food Sci., 1962, 27, 537). Each fraction is then resolved into its constituents using column chromatography. Using these techniques, Curl and his co-workers have surveyed the carotenoids present in a wide variety of fruits (J. Food Sci., 1961, 26, 442; Food Res., 196o, 25, 19o, 670; 1959, 24, 413; 1957, 22, 63; J. agric. Food Chem., 1956, 4, 156; 1954, 2, 685; and refs. cited above). Some of the many minor components usually detected in a detailed analysis of the carotenoid mixture obtained from a natural source are probably artefacts [e.g., stereoisomers of the naturally-occurring form (p. 250) or oxidation products] formed during the isolation procedure (cf. Bickol~ et al., loc. tit., who found that a solution of neoxanthin left for two days in the

242

THE CAROTENOID GROUP

7

dark at 250 produced six new pigments; four other xanthophylls behaved similarly). It is difficult to overemphasise the usefulness of column chromatography to carotenoid chemistry; it was introduced in 1931 by R. K u h n and E. Lederer (Ber., 1931, 64, 1349). A wide variety of adsorbents and solvents has been used. Alumina, magnesia, and calcium hydroxide, with hexane, hexane-ether, hexane-acetone (except on strongly basic adsorbents) or petrol-benzene mixtures as solvent, are frequently used to separate carotenes and the less polar xanthophylls. For the more polar xanthophylls powdered sucrose, cellulose, zinc carbonate, or calcium carbonate are preferred; astacene, which forms a salt with basic adsorbents, can be conveniently chromatographed on sucrose from benzene-petrol. Alumina packings of various activities, as prepared by the controlled deactivation of active alumina with methanol or water (cf. T. I. Williams, "Elements of Chromatography", Blackie & Son, London, 1954), can be used for most of the less polar carotenoids. Stereoisomers of a single carotenoid can often be resolved (cf. p. 251). Carotenoid epoxides (p. 293 ) undergo gradual rearrangement and decomposition on alumina; lime or magnesia columns are usually used for these compounds. The effectiveness of functional groups on the degree of adsorption generally decreases in the order hydroxyl group, carbonyl group, ester or ether function, double bond. However, the exact sequence obtained on chromatographing a carotenoid mixture depends on the particular combination of adsorbent and developing solvent used (Strain, J. Amer. chem. Soc., 1948 , 70, 588). F u r t h e r details and references are given by Goodwin (1955, loc. cir.) and Strain (oJ). cir., 1958 ). The chromatography of carotenoids is facilitated by their ready visual detection; the eluate is conveniently monitored by its light absorption properties. The intense colour of most carotenoids also makes them particularly amenable to thin layer (T.L.C.) and paper chromatographic separations, no detecting agent normally being necessary. T.L.C. has been used to resolve (on "normal-phase" silica gel, magnesia, alumina, etc. plates) mixtures of carotenoids (E. Demole, Chromat. Rev., I959, I, I ; O. Isler et al., Chimia, I96I, I5, 208; H. R. Bolliger et al., ibid., I964, I8, I36; E. C. Grob and R. P. Pflugshaupt, Helv., I962, 45, 1592, K. Randerath, "Thinlayer Chromatography", Verlag-Chemie, Weinheim, I966, pp. I82, I89) and of vitamin A stereoisomers (Isler et al., Helv., I962, 45, 548) into their components. Some loss, by decomposition on the plate, usually occurs; this can be minimised by using cellulose layers (H. ,4. W. Schneider, J. Chromatog., I966, 2I, 448). "Reversed-phase" (paraffin off on silica gel) plates give better separations in the polyene aldehyde series (,4. Winterstein et al., Ber., I96o, 93, 295I; cf. also

3

STRUCTURE

243

Randerath, loc. cit.) and are particularly useful for highly oxygenated compounds, such as astacene and fucoxanthin (J. B. Davis, unpubl.). Weakly coloured (pale yellow) compounds are readily detected by their UV fluorescence (vitamin A, phytofluene) or by using antimony trichloride, permanganate, etc., sprays. Winterstein and B. Hegedi~s (Chimia, 196o, 14, 18) have described a sensitive detecting agent for polyene aldehydes (o. I /zg detectable). Good separations can also be effected on circular filter papers impregnated with kieselguhr (~ 20% w/w) using acetone-petrol mixtures as developers; stereoisomers can be cleanly separated (A. Jensen and S. L. Jensen, Acta Chem. Scand., 1959, 13, 1863). Reversed-phase papers have been used in the vitamin A series (F. B. Jungalwala and H. R. Cama, J. Chromatog., 1962, 8, 535), and two-dimensional paper chromatography has been used for mixtures containing both carotenoids and chlorophylls (S. W. Jeffrey, Biochem. J., 1961, 8o, 336). In all three methods, the colouring matters are easily recovered from the coloured spot or zone by elution with acetone or methanol and spectral identification and/or assay can then be carried out. 3. Structure and synthesis

(a) Determination of structure The structures of m a n y of the carotenoids have been elucidated by the application of the following general techniques. (i) Measurement of hydrogen absorption on microhydrogenation gives the number of carbon-carbon double bonds (carbonyl groups are also saturated but more slowly); coupled with analysis of the original colouring matter (which, however, is rarely able to distinguish unequivocally between e.g., H54 and Hse), this also indicates the number of rings present. (ii) The visible light absorption spectrum gives an indication of the chromophore (see p. 252). (iii) Zerewitinoff and Zeisel determinations give the number of hydroxyl and methoxyl groups. (iv) Oxidative degradation with ozone and with permanganate indicates the type of ring system(s) present. Thus carotenoids containing unsubstituted a- and fl-ionone end-groups give isogeronic and geronic acids, respectively, (see Vol. ID, p. 267), on ozonisation: Me

Me

Me

Me

Isogeronic acid

Me Me

Me

Me

Ger'onic acid

The presence and position of oxygen substituents can be deduced from oxidation with permanganate" carotenoids with unsubstituted a - o r fl-ionone

244

THE CAROTENOID

type end-groups dimethylsuccinic oids carrying an or XIII + XIV Me

GROUP

both give a mixture of aa-dimethylglutaric (XIII), aa(XIV), and dimethylmalonic (XV) acids, whereas carotenoxygen function at C(,), C(3) or Ca) give XV, XIV + XV + XV, respectively (Scheme I).

Me

Me

Me

Me

Me

CO2H Me

7

Me

OaH

Me

CO~ CO#t

C%H

Me

/ (~rrr)

CZ~)

(X~)

Scheme i.

In the earlier work (P. Karrer et al., Helv., 193o, 13, lO84) the acids were separated by fractional crystallisation and so a large quantity of carotenoid material was needed. Less is needed if the acids are separated by paper chromatography and identified by carrying out mixed chromatograms with authentic specimens (37. F. tIotyer and B. C. L. Weedon, Chem. and Ind., 1955, r219; cf. L. Cholnoky and J. Szabolcs, Experientia, 196o, 16, 483). Alternatively, the acids may be identified, and their relative yields determined, by gas-liquid chromatography of their methyl esters. (v) The number of isopropylidene groups is determined by ozonisation to acetone (R. Kuhn and H. Roth, Ber., 1932, 65, 1285); isopropyl, c~-ionone, and/~-ionone residues also give small amounts ( ~ o. I - o. 2 mole) of acetone (Kuhn and Roth, loc. cit. ; L. Zechmeister and W. A. Schroeder, Arch. Biochem., 1943, i, 231); --CM%OH and--CM%OMe residues yield ~0"3 mole (cf. S. L. Jensen, Acta Chem. Scand., 1959, i3, 842). (vi) Side-chain methyls are determined by oxidation with chromic acid to acetic acid (Kuhn and Roth, Ber., 1933, 66, 1274); the geminal methyl groups yield no acetic acid and the olefinic methyls usually yield rather less than the theoretical amount. [The substitution j~attern of the central polyene chain in lycopene (and thence, mainly by analogy, in other cartenoids) was first elucidated by comparing its perhydro-derivative with the totally synthetic material (p. 266), and by relating lycopene itself to bixin and thence to crocetin (whose structures were again proved by comparing their perhydro-derivatives with totally synthetic samples, pp. 326 et seq.) ]. (vii) Vitamin A activity: carotenoids possessing an unsubstituted fl-ionone ring show high vitamin A activity; most, but not all, others do not (p. 283).

3

STRUCTURE

245

(For further details of these methods, cf. Karrer and E. Jucker, "Carotenoids", Elsevier, Amsterdam 195o, and for an example of their application, see W. J. Rabourn and F. W. Quackenbush, Arch. Biochem. Biophys., r956 ,

6I, III). Most of the above methods require relatively large amounts of material and have been used for the more readily available carotenoids. However, even when only a small amount (e.g., I mg.) of a carotenoid is available, some idea of its structure can be gained from small-scale tests. Thus the type of functional groups present can often be inferred from its behaviour on a chromatogram or on countercurrent distribution (pp. 242, 241) and from its partition behaviour (both before and after saponification), the epiphase: hypophase ratio (p. 235) being compared with the values obtained with carotenoids containing various (known) functional groups (F. J. Petracek and L. Zechmeister, Anal. Chem., 1956, 28, 1484; N. I. Krinsky, Anal. Biochem., 1963, 6, 293; C. Subbarayan er al., ibid., 1965, 12, 275). The light absorption spectrum can still be obtained, and the presence of a polycis compound can be recognised by adding iodine (see p. 255) and of an epoxide (p. 293 ) or allylic hydroxyl group (p. 264) by adding acid to the solution in the cell. The presence of a carbonyl group conjugated with a polyene chromophore can be detected spectrally (p. 254), through oximation (P. 254), or by reducing with borohydride and showing that the product contains (cf. p. 264 et seq.) an allylic hydroxyl group. Polyene aldehydes and polyene ketones can be differentiated according to their rate of reduction by borohydride under standardised conditions (cf. A. L. Curl, J. Food Sci., 1962, 27, 537). An isolated carbonyl group can be detected by observing the effect of borohydride reduction on chromatographic behaviour. In recent years increasing use has been made of infra-red (I.R.) and proton magnetic resonance (P.M.R.) methods which, being non-destructive and requiring only small samples, are particularly suited to the carotenoid field: cf. p. 256 el seq. The more recent publications of S. L. Jensen and co-workers serve to illustrate the use of small-scale techniques of structure determination (cf. e.g., p. 307 et seq.).

(b) Synthesis Since 195o, when the first total syntheses of fl-carotene were announced (see p. 268), many of the carotenoids have been synthesised, some of them by several different routes. The field of carotenoid synthesis is now very extensive and only a few of the more important procedures that have been used can be included in the present account. (A comprehensive review is

246

THE CAROTENOID

GROUP

7

given by 0. Islet and P. Schudel in "Advances in Organic C h e m i s t r y " , Vol. 4, Interscience, 1963). The first syntheses of several of the carotenoids (e.g. fl- and a-carotenes, pp. 268, 271) were accomplished by Karrer and his co-workers who used the C16 + C8 + C10--~ Cao principle. The C8 unit is a diketone (see XLVIII, p. 269), originally obtained in low yield from glyoxal and acetoacetic acid (Helv., 1949, 32, I934), but later obtained in rather better yield from acetylene (Weedon and co-workers, J. chem. Soc., 1952, 4089)*. The final step using this route is a low-yield dehydration of a C40 tetraol to the corresponding polyene. Methods have, however, now been developed whereby the polyene chain can be extended by two or three carbon atoms at a time, the double bonds being introduced concomitantly rather than by multiple dehydration at the end of the sequence. One of these, the vinyl ether synthesis (cf. Islet et al., Helv., 1956, 39, 249), is applied to aldehydes. The aldehyde XVI is acetalised (ethyl orthoformate) and the acetal is treated with ethyl vinyl ether (CH,= CHOEt) in the presence of zinc chloride" acid hydrolysis of the product gives the aldehyde XVlI. If this treatment is then repeated using ethyl propenyl ether (MeCH-CHOEt) a side chain methyl is inserted, giving XVIII** : R

O

~

R

------"

R

,~

----,.-

CH(CEt) 2

CH(OEt) 2

(X3ZI)

OEt

R/~//0

= R ~ O

(R'~7IT)

(R'~/m')

In this way the fl-C14-aldehyde (XIX) used in vitamin A synthesis (see p. 285) can be converted (Islet el al., Helv., 1956, 39, 249) into the fl-C19aldehyde (XX), used in the synthesis of fl-carotene and kryptoxanthin" O

(IIX9

~

~

O

~X~

* For improved syntheses of this potentially useful intermediate, see K. Eiter et al., Ann., I965, (184, I4. ** For a method of converting compounds of the type R...~..~O directly into R ...#...#...#...~ t w O see J. Redel and J. Boch, Compt. rend., 1964, 258, 184o, and for another route to aldehydes of the type discussed here, cf. M. Julia et al., Bull. Soc. chim. Ft., 1966, 728.

3

247

SYNTHESIS

The central C5 unit of the carotenoid skeleton can be added (Isler et al., ibid., 1959, 42, 847) by condensation of XX (Grignard reaction or lithamide in ammonia) with the acetal XXI, which is itself prepared from the enol ether of methylmalondialdehyde (XXII) as shown (Scheme 2). OH

~

(Z~:) + (ZZ:I)

CH(OE:{}z

(C~)

~

etc.

Scheme 2. The C2~-aldehyde (XXIII) so formed can now be submitted to chain extension with alternately ethyl vinyl ether and ethyl propenyl ether so as to obtain the C27, C3o, C~,, C35, C3v and C,0 polyene aldehydes (Islet et al., ibid., 1959, 4z, 854). [An analogous series of polyene esters has been synthesised (Islet et al., ibid., 1959, 42, 864) by condensing the aldehydes with the alkylidene phosphoranes (Wittig reagents), Ph3P=CMe.CO2Me or PhzP= CH. CO2Me (cf. e.g., synthesis of torularhodin, p. 325) ]. Several of the polyene aldehydes have been used in syntheses of C40 carotenoids by adding the appropriate end-group (frequently by the Wittig reaction) (cf. pp. 272, 273, 280). The central triple bond is frequently left in the molecule until the last stage (acetylenic derivatives are more soluble and better yields are occasionally obtained). Selective reduction of the triple bond using Lindlar catalyst (H. Lindlar, ibid., 1952, 35, 446) and stereoisomerisation of the I5,I5'-cis-carotenoid so produced by refluxing in petrol (Isler et al., ibid., I959, 42, 84I) or iodine catalysis (p. 25I) affords the all-trans compound. A two-stage Wittig synthesis has been employed in the construction of unsymmetrical carotenoid molecules (e.g. 6-carotene) ; a polyene dialdehyde is treated with one mole of the Wittig salt derivative of one end group under carefully controlled conditions, the desired I ' I addition product is isolated chromatogxaphically, and is then linked with the other end-group using a

248

THE CAROTENOID GROUP

7

second Wittig reaction (R. Riiegg, Weedon et al., J. chem. Soc., I965, 2o19). For additional examples, cf. pp. 273, 279, 305, 308. Several of the symmetrical carotenoids have been synthesised using routes based on the C=o-dialdehyde, crocetindial (XXIV) (i.e. C10 + C20 + C10 ~C4o: see e.g., pp. 267, 278, 318):

~

0

Crocetindia[ ~ )

This compound already contains most of the to be prepared and can be obtained in good obtained (Isler et al., Helv., 1959, 42, 847) acetylene X X I using sodamide in liquid E t O C H ~

CH(OEt)2

chromophore of the carotenoid yield from the product XXV by condensing X X l I with the ammonia. The C10-dialdehyde

EtO~ ~ HC(O~,,,/~

E,o 7

~ .v

OH (ZX3Z)

~ -.~v

/~.OEt "T

-oE,

---"

(ZX2D

0 ~

~

0

--" (ZZ]~

(X'xw)

diacetal (XXVI) is converted via the C14- (XXVlI) into the C20-dialdehyde* (XXIV), using first the vinyl ether and then the propenyl ether synthesis and, finally, selective reduction of the triple bond (cf. above) (Islet et al., ibid., 1956, 39, 463) 9 The C19 + C2 + C19 scheme has also often been used, two C19 units (aldehydes) being linked by a Grignard reaction with acetylenedimagnesium bromide (cf. pp. 270, 298, 319). Cis double bonds can be introduced into the polyene chain by partial reduction of the corresponding acetylenic compound as above (for examples, see p. 285), or through a Wittig reaction (G. Pattenden et al., Chem. Comm., 1965, 347) thus: R'CH'-'-PPhs

* For syntheses of the symmetrical, fully unsaturated dialdehydes of the type XXIV containing 24, 3o, 34, and 4~ (with a O--C. (C--C)j 5. C =O chromophore) carbon atoms, and of the corresponding diacid diethyl esters, cf. Isler et al., Helv., 1966, 49, 369.

4

GENERAL

PROPERTIES

249

Several of the carotenoids have been prepared from other (synthetic) carotenoids by "partial synthesis"; for example: the epoxides from the corresponding parent carotenoids by treatment with per-acid (p. 293); canthaxanthin from fl-carotene or I5,I5'-dehydro-fl-carotene (using Nbromosuccinimide/acetic acid followed by an Oppenauer oxidation); echinenone from fl-carotene (boron trifluoride complex + water, followed by Oppenauer oxidation); astacene from canthaxanthin (oxygen/potassium tert-butoxide); bisdehydrolycopene from lycopene; eschscholtzxanthin from zeaxanthin (N-bromosuccinimide), etc.; details of each of these syntheses are given under the appropriate carotenoid. fl-Carotene, canthaxanthin, fl-apo-8'-carotenal, ethyl fl-apo-8'-carotenoate, and vitamin A are now all synthesised on an industrial scale, mainly for use as food colourants and/or as provitamins A (cf. Islet et al., Chimia, 1961, 15, 208). In addition, certain carotenoids could probably be produced on a large scale microbiologically. For example, the organism Blakeslea trispora can be cultivated in such a way (nutrient medium containing compounds known to stimulate carotenoid formation, optimum growth period, etc.) that it produces relatively large quantities of a single carotenoid (fl-carotene), and this could be extracted with solvent and the solution either processed to fl-carotene itself or evaporated to give a crude concentrate with high provitamin A activity (cf. A. Ciegler, Adv. appl. Microbiol., 1965, 7, 18; C.A., 1966, 64, 7328h, 2o597c). 4. Properties of carotenoids (a) General properties; cis-trans isomerism The numerous conjugated double bonds in a carotenoid are responsible for its colour and its tendency to undergo atmospheric oxidation, and allow exceptional opportunities for cis-trans isomerism. Even pure crystalline carotenoids deteriorate on keeping in air, the rate of oxidative destruction depending markedly on the actual compound. Carotenoids containing a conjugated keto group tend to be more stable than the hydrocarbons. The number of allylic methylene groups in a carotenoid may be an important factor" the carotenoid precursors (phytoene, etc.: p. 335), which contain many allylic methylenes, are exceptionally sensitive to aerial oxidation. Once sealed under vacuum, most pure carotenoids can be stored unchanged for many years. Most carotenoids can be stored in non-polar solvents at o ~ without extensive loss, although they do tend to undergo trans ~ cis isomerism (see below). At ioo ~ lycopene is quite rapidly destroyed by oxygen even in solution (25 % lost in 3 hr.) especially in the presence of copper ions (88% lost) (E. R. Cole and

250

THE CAROTENOID GROUP

7

N . S. tCapur, J. Sci. Food Agr., 1957, 8, 36o); in benzene at 5 o~ fl-carotene gives first a m i x t u r e of its epoxides and their furanoid derivatives (cf. p. 293) and semifl-carotenone (p. 26o), b u t these are soon destroyed (R. F. Hunter and R. M. Krakenberger, J. chem. Soc., 1947, I, cf. also H. v. Euler et al., Ber., 1929, 62, 2445).

Carotenoids are sensitive to acids to varying degrees. Carotenes are stable to dilute acids and even concentrated hydrochloric acid causes little chemical change (but does promote stereoisomerisation: see below). However, xanthophylls, especially those containing allylic hydroxyl groups or epoxide rings, undergo irreversible chemical changes with acids (pp. 264, 293 ). Carotenoids give intense blue colours when treated with antimony trichloride in chloroform (the Cart-Price reaction); the absorption band(s) giving rise to the colour (usually in the 550 to 700 m# region) has been used, particularly in the vitamin A field, as a means of detecting and estimating polyenes in crude extracts, fl-Carotene dissolves in strong acids to give an ionic (electrically-conducting) complex which if basified immediately liberates the carotene; irreversible changes take place on standing (A. Wasserman, J. chem. Soc., 1954, 4329; F. Kdr6sy, Experientia, 1955, ix, 342; cf. P. E. Blatz and D. L. Pippert, Tetrahedron Letters, 1966, 1117). In 1935-1938 it was observed that on repeated chromatography of pure 15-carotene or on prolonged storage of its solutions, a second colouring matter ("pseudo-c~-carotene") was formed (A. E. Gillam and M. S. El Ridi, Biochem. J., 1936, 30, 1735; L. Zechmeister and P. Tuzson, ibid., 1938, 32, 13o5). It was soon found that other carotenoids behaved similarly and it became apparent that they were all undergoing spontaneous trans--,, cis isomerism (cf. A. Polgar and Zechmeister, J. Amer. chem. Soc., 1942, 64, 1856). It is now recognised that this is general (cf. Zechmeister, "Cis-trans Isomeric Carotenoids, Vitamins A, and Arylpolyenes", Springer, Berlin, 1962; Fortschr. Chem. org. Naturst., 196o, 18, 223). The total number of stereoisomeric forms theoretically possible for a given carotenoid (assuming that one or more of any of the acyclic double bonds may be either cis or transl is readily calculable, the values for fl-carotene, a-carotene, and lycopene being 272, 512, and lO56, respectively (Zechmeister, 196o, loc. cir., pp. 232-5). As was first pointed out by L. Pauling (in I939), two types of cis double bond can be distinguished in a carotenoid molecule- unhindered (a) and hindered (b):

. ~'C C~\H HI (a)

~C

/H

\ CHsH (b)

/

C~

4

GENERAL PROPERTIES

251

Of the nine acyclic double bonds in, for example, fl-carotene (I), five are unhindered (the four carrying methyl substituents and the central double bond) the remaining four being hindered. Considerable steric hindrance opposes the formation of a hindered cis bond. The number of possible stereoisomers in the above examples containing only unhindered cis bonds is 20, 32, and 72, respectively. Carotenoids in which all acyclic double bonds have the trans configuration (all-tram carotenoids) are readily c o n v e r t e d into a m i x t u r e of stereoisomers in which one or more of these double bonds is cis, by (a) exposure to light in presence of iodine which acts catalytically; (b) heating in a hydrocarbon solvent (in the dark); (c) briefly melting crystals of the c o m p o u n d in vacuo; (d) prolonged contact with an active surface (e.g., active alumina); (e) strong illumination in the absence of catalysts (and air); (f) t r e a t m e n t with acid. The q u a n t i t a t i v e composition of the m i x t u r e of stereoisomers produced from a carotenoid depends on the m e t h o d used. Stereoisomers containing hindered cis bonds are only very exceptionally produced by the above procedures (for examples, see Zechmeister, 1962, loc. cit., pp. 91, 125): however, such c o m p o u n d s can be obtained synthetically by partial reduction of the corresponding acetylenes (cf. p. 247, and see P. Karrer et al., Helv., I952, 35, 185o; 1953, 36, 828; 0. Islet et al., ibid., 1957, 40, 1256; 1962, 45, 517). A hindered cis isomer of v i t a m i n A aldehyde is involved in t h e visual process (cf. p. 289). Of the above methods, (c), (e) and (f) frequently also cause some irreversible destruction [(c) and (e)] or chemical change (f) of the carotenoid. Most stereoisomerisation work is carried out using method (a); for this, a solution of the carotenoid in hexane or benzene containing a trace ( ~ 1% on the amount of carotenoid) of iodine is exposed to artificial light for a few minutes (Zechmeister, 196o, loc. cit., p. 272). Usually about one half of the product consists of the all-trans form, the remainder being a mixture of cis forms. The mixture of stereoisomers can usually be resolved into its constituents by chromatography: the cis isomers commonly appear below the all-trans on the column but occasionally some appear above. The isomers are usually named according to the sequence in which they appear, e.g . . . . neo-fl-carotene U, alltrans, neo-fl-carotene A, neo-B, neo-C . . . . (from top to bottom of the column). Treatment of any one of the cis isomers so produced with iodine in light as before gives the same equilibrium mixture of stereoisomers as was obtained from the all-trans form. The rate at which a cis isomer undergoes stereoisomerisation depends on the number and type of cis bonds it contains and the isomerisation method used. A carotenoid containing a hindered cis bond is, once formed, relatively stable to heat but is very sensitive to iodine in light, giving the equilibrium mixture much faster than either the all-trans or any of the unhindered-cis forms of the same carotenoid; naturally-derived polycis carotenoids, which

252

THE C A R O T E N O I D GROUP

7

contain several unhindered-cis bonds, e.g. prolycopene (p. 267), pro-~-carotene, b e h a v e similarly. B y t h e controlled exposure of a n a t u r a l polycis-lycopene to light (in the absence of a catalyst), E. F. Magoon and Zechmeister (Arch. t3iochem. Biophys., 1957, 69, 535) o b t a i n e d a series of cis-lycopenes corresponding to the stepwise stereoisomerisation of the various cis bonds originally present. In addition, m a n y cis-lycopenes h a v e been isolated from n a t u r a l sources (mainly polycis: p. 267) and others h a v e been prepared from all-trans-lycopene b y applying t h e

stereoisomerisation procedures above or by total synthesis, so that in all 41 isomers of lycopene containing one or more cis bonds have now been recognised (for list, cf. Zechmeister, 196o, loc. cit., p. 289). The all-tram form of a carotenoid is generally more stable than any of its cis derivatives: it can be crystallised unchanged whereas a chromatographically-pure cis-carotenoid frequently yields crystals which contain some of the all-tram form. It also has a higher melting point (and is less soluble) than any of its cis isomers. As already mentioned, carotenoids undergo trans-cis isomerism when stored in solution even in the dark (ct. p. 250). With most carotenoids this is a relatively slow process but spirilloxanthin and rhodovibrin provide notable exceptions (Zechmeister et al., Arch. Biochem., 1944, 5, 243; S. L. Jensen, Acta Chem. Scand., 1959, 13, 2143). As expected, the optical rotation of the all-trans form of an optically active carotenoid differs from that of its cis isomers (e.g., all-trans-zeaxanthin [~ -- - - 4 2 . 5 ~ neozeaxanthin A, + 12o 0. other examples are quoted in Zechmeister 196o, loc. cir., p. 240 ). Regarding the spectral properties of cis isomers, see p. 254 et seq.

(b) Physical properties (i) Visible and ultra-violet absorption spectra* All carotenoids show intense absorption in the visible region of the spectrum. The shape of the absorption curve as well as the position of its maxima depends on the precise nature of the chromophore, so that examination of the absorption properties of a new carotenoid usually provides a useful indication of the chromophore present. The shape of the curve depends on the conformation of the chromophore, an all-tram planar chromophore showing the greatest degree of fine structure - - t h r e e distinct peaks in the visible region separated by ~ 20 to 40 m/~; of these, the central peak is usually the most intense and is denoted in heavy * See also Vol. IA, pp. i 13 et seq. for basic theory of visible and ultra-violet absorption spectra.

4

PHYSICAL PROPERTIES

253

type later on (see p. 266 et seq.), the longest wavelength peak is rather less intense, and the third (shortest wavelength) peak is weaker and less well defined. Any carotenoid in which the chromophore does not extend into a ring (and is not terminated by a carbonyl g r o u p p f o r which see below)gives this type of spectrum (examples" lycopene, ~-carotene, violaxanthin, auroxanthin). As the length of the polyene chain increases so the absorption bands move steadily to longer wavelengths (and the colour of the compounds changes from yellow to red), although the increment (per C--C) gradually falls off (e.g. the central peak in the spectra of the acyclic hydrocarbons containing 7, 9, II, 13 and 15 conjugated double bonds occurs, in hexane, at 4Ol, 440, 472, ~ 500, and 51o m/~, respectively)" simultaneously the intensity of absorption steadily increases (Smax. for the first three in the above series is 138,ooo, 16I,ooo and 186,ooo, respectively). The spectra of carotenoids (e.g. >,-carotene) with a chromophore which extends into a fl-ionone ring differ from the above in three respects" (i) decreased fine structure; (ii) decreased intensity; (*ii) bands at shorter wavelength than the analogous acyclic carotenoid" a ring double bond has only about one third of the effect of an acyclic double bond (e.g. ~,-carotene with a nominal I I double bonds has absorption bands at the wavelengths expected for a hydrocarbon with "1o'4 conjugated double bonds"). These effects are ascribed to steric hindrance between the ring methyls and the hydrogens on C(7) and/or C(8) preventing the ring double bond being trans to and coplanar with the polyene chain (cf. p. 236) so that maximum overlap of ~-orbitals is prevented. If the chromophore is terminated at both ends by a ring double bond (as in fl-carotene) the above effects are twice as marked (the short wavelength peak now being just an inflexion). In contrast, retrodehydro-fl-carotene (XXXVI, p. 26I), which has an approximately planar chromophore (J. Dale, Acta Chem. Scand., 1954, 8, I235), shows good fine structure and intense absorption (Smax. = I65,ooo) (Zechmeister and L. Wallcave, J. Amer. chem. Soc., 1953, 75, 5341) 9 Further loss of fine structure occurs on extending the chromophore further into the fl-ionone ring, as in the (hypothetical) sequence"

O

/~-Carotene

~'max 451 m/,

3 , 4 : 3 ~. 4 ' - Bisdehydro-/5 carotene ~ma,~472 mp

O

4 . 4 ' - Dioxo-/~ carotene (canthaxanthin) )]' max 4 6 6 m~

Astacene

~'ma• 477 rr~

both the bisdehydro-/5-carotene and canthaxanthin give only a single broad (but not quite symmetrical) peak" astacene gives a symmetrical broad peak.

254

THE

CAROTENOID GROUP

7

Renierapurpurin, renieratene, and isorenieratene (see p. 277 ) absorb {in carbon disulphide) at ~ 26, 33 and 46 m/~ shorter wavelength than would be expected if the benzene rings were coplanar with the polyene chain, this being prevented in this series to a progressively greater degree as the number of methyl substituents on the benzene rings ortho to the polyene chain is increased. The spectra of all carotenoids are solvent-dependent. Representative values for the position of the central peak in a carotenoid absorbing at 51o m/z in carbon disulphide are: hexane, 476; ethanol (except for polyene ketones: below) or ether, 478; chloroform, 488; and benzene, 49 ~ m~. A carotenoid will therefore be more deeply coloured in carbon disulphide than in other solvents. However, extinction coefficients are usually highest in hexane and ethanol and relatively low in carbon disulphide. Chromophores terminated by a carbonyl group show a characteristic solvent effect: whereas these chromophores (XXVlII), if planar, give rise RI

R' (X~Tr a )

(~c9'rn'b)

to a curve with good fine structure in hexane solution, in ethanol the curve degenerates to a single broad peak with a weak long wavelength shoulder, the centre of the absorption band simultaneously moving 5-15 m/~ to longer wavelength (cf. Polgar and Zechmeister, J. Amer. chem. Soc., 1944, 66, 186; A. L. Curl, J. agric. Food Chem., ~962, io, 504). This provides a useful small-scale method of detecting a conjugated carbonyl group in a carotenoid of unknown structure; (in addition, oximation of a conjugated carbonyl causes a spectral shift of 5-1o m/~ to shorter wavelength: R. Kuhn and H. Brockmann, Ber., :1933, 66, 828; I. M. Heilbron and B. Lythgoe, J. chem. Soc., 1936, 1376 ). Planar chromophores terminated b y - - C O 2 H normally show some fine structure in both hexane and ethanol. The absorption curve of the all-tram form of a carotenoid has its peaks at longer wavelengths, has higher extinction coefficients, and shows more fine structure than the curve obtained from any of the cis isomers. The first of these generalisations, however, holds neither for vitamin A, nor for polyenes lacking methyl substituents, cis compounds being known which absorb at longer wavelengths (but having lower extinction coefficients) than the all-tra~s form (C. D. Robeson et al., J. Amer. chem. Soc., 1955, 77, 4111; E. R. H. Jones et al., Chem. and Ind., 1956, 928; L. Crombie et al., J. chem. Soc., 1957, 2754).

4

PHYSICAL

255

PROPERTIES

In addition, the spectra of many of the cis forms of a carotenoid contain a peak (the "cis-peak") in the ultra-violet region which is absent from the curve of the all-lrans form. The cis-peak has a well defined location, occurring (in hexane) at 142 + 2 m# shorter wavelength than the longest wavelength band of the all-tram form in carotenoids with I O - l l conjugated double bonds, the separation becoming progressively less as the chromophore is shortened. The cis-peak is always less intense than the main (visible)

M Me

Me,

Me

Me

M

Me Me

Me

Me

(xx'~)

absorption band reaching its greatest (relative) intensity in central-cis carotenoids (which have markedly "bent" chromophores (cf. XXIX) where it commonly has 50 to 7 ~ % of the intensity of the main maximum. Linear carotenoids (all-tram carotenoids) and also those poly-unhinderedcis carotenoids (including natural polycis carotenoids) with chromophores having an approximately linear overall shape (cf. XXX) show no cis-peak effect. (In general, the intensity of the cis-peak is roughly proportional to the square of the distance between the centre of the conjugated system and the mid-point of the straight line between its two ends). The main (visible) absorption band of a polycis carotenoid occurs at much shorter wavelength than that of the corresponding all-tram compound and shows little fine structure; iodine isomerisation (p. 251) (polycis--+ cis/trans equilibrium mixture) produces a marked shift (commonly of 30 to 50 m/~) to longer wavelengths (a pale yellow solution of a polycis carotenoid turns deep orange in a few seconds on illumination in the presence of iodine): iodine isomerisation of an all-tram carotenoid produces a relatively small shift to shorter wavelengths and a loss of fine structure. Carotenoids containing a hindered cis bond give spectra which show little or no fine structure in the

256

THE CAROTENOID

GROUP

7

main (visible) absorption band and which usually have cis peaks; with iodine they behave in the same way as polycis carotenoids. For typical absorption curves and extensive references, see Zechmeister, "Cis-trans Isomeric Carotenoids, Vitamins A, and Arylpolyenes", Springer, Berlin, 1962; Fortschr. Chem. org. Naturst., 196o, 18, 223. For theories of carotenoid spectra, see Zechmeister, 196o, loc. cit., p. 255; J. N. Murrell, Acta Chem. Scand., 1961, I5, 1783; J. Dale, ibid., 1954, 8, 1235; H. Suzuki and S. Mizuhashi, J. phys. Soc. Japan, 1964, 19, 724; C.A., 1964, 6I, 38o8h. Spectra measured at - - I 9 o~ show increased fine structure and intensity, and a shift ( ~ 20 m/~) to longer wavelengths (E. S. Miller, Plant Physiol., 1934, 9, 179; G. Wald et al., Nature, 1959, 184, 617, 620).

(ii~ Infra-red spectra Normal carotenoids give a strong band near 964 cm. -1 (C--H out-of-plane deformation of trans C H - C H ) , whereas two bands appear in the spectra of carotenoids of the retro type (~ 975, 960 cm.-1), and also in central-cis carotenoids (barely resolved) (H. H. Strain et al., J. org. Chem., 1961, 26, 5061 ; O. Islet et al., Helv., I956, 39,454, 249 ; M. A khtar and B. C. L. Weedon, J. chem Soc., 1959, 4058). Carotenoids containing conjugated keto-groups (e.g. capsorubin) give three strong bands in this region (near lOO5, 980 and 97 ~ cm. .-1) (C. K. Warren and Weedon, J. chem. Soc., 1958, 3972). Cis double bonds can be detected, and differentiated: an unmethylated cis double bond (--CH ~ C H w ) gives a strong peak near 780 cm. -1 (C--H def. of cis CH =CH) whilst a methylated cis double bond ( ~ C M e ~ C H - - ) gives rise to a small but distinct band at 138o cm. -~ (C--H def. of H ~ C . C - - ) (K. Lunde and Zechmeister, J. Amer. chem. Soc., 1955, 77, r647). Carotenoids containing cyclohexenone end-groups (e.g. canthaxanthin)(VCmHxc]' of carbonyl group in the range I65o to 1655 cm.-1 with e per C = O of ~ 450) can be distinguished from those (e.g. capsorubin) containing non-cyclic conjugated keto-groups (.c-c23 in the range 166o to 167o cm. -~" e per C=O, ~ 200)9 In addition , Vma x the latter also give two strong C - C bands (near 158o and 154o cm.-1). The number of keto-groups can be estimated from the intensity of the C - O band (Warren and Weedon, J. chem. Soc., 1958, 3972, 3986). Whether the OH or OMe group in hydroxy- and methoxy-carotenoids is attached to a secondary [C~3)] or tertiary [C(~)] carbon can be inferred from the precise position of the C ~ O band (S. L. Jensen, Acta Chem. Scand., 1959, 13, 842, 2142; 1963, x7,489,5oo) ; the groupings R . C H O H . C - C . and R-CHOH.CH~.C=C- can similarly be distinguished (C. Bodea et al., Ann., 1963, 666, 189). (iii) Proton magnetic resonance (P.M.R., N.M.R.) The methyl groups in carotenoids are attached to fully substituted carbon atoms and give singlets [broadened in the case of --CMe = C H - - methyls by

4

PHYSICAL PROPERTIES

257

weak (allylic) coupling]. The olefinic protons give complex patterns in the 3"5-5 region due to extensive coupling. Typical z values are (L. M. Jackman et al., J. chem. Soc., 196o, 2870): 8"41 Me

8-20 Me

8.97 ~ Me Me

8"03 Me

803 Me

8.35

8"81

9-06,8-90

Me Me

8"89,8"84

Me Me

Me

0

8-21 0

~

8"86

__

0 8-57

Thus, "in-chain" methyls (z 8"o3) can be distinguished from "end-ofchain" methyls (3 8.20 for the acyclic type, z 8.28 for the "/5 end-group" type, etc.) and many of the known carotenoid end-groups can be readily detected and differentiated: the furanoid oxide end-group is characterised by a two-proton peak at ~ 4" 85 due to the C~) and C(8) protons, which happen to be accidently coincident. Methoxy (3 ~ 6-75) and aldehyde (3 ~ 0"5) functions are readily detected. P.M.R. spectroscopy has been instrumental in the elucidation of the structures of spirilloxanthin (p. 3o8); capsanthin, capsorubin, and kryptocapsin (pp. 322-325) ; neurosporene, ~-carotene, phytofluene, and phytoene (cf. p. 335); chlorobactene (p. 279); pigments " Y " , "R", "OH-Y", "OH-R", and "P 518", and chloroxanthin (pp. 31o-311); 3,4-dehydrorhodopin (p. 307); and others. The stereochemistry of natural bixin has been elucidated through the application of this technique (p. 327). The position of the C(13) methyl in 5 stereoisomers of vitamin A (p. 282) varies from z 8.12 to 8- 30 depending on tile stereochemistry of the CO), C(n ) and C(13)double bonds (C. von Planta, Vitamins and Hormones, 196o, 18, 323). The P.M.R. spectra of many of the carotenoids, and of the vitamins A, have been measured either in conjunction with structure determination (cf. the examples noted above) or for record purposes; appropriate references are given in the sections describing the individual compounds (p. 266 et seq.). (iv) Mass spectrometry This technique has afforded the first reliable method of determining molecular weights. It has also been used to determine molecular formulae (by precise determination of molecular weight) and to detect, and estimate the number of, OH groups (presence of M-I8, M-(2 • I8), etc. peaks), acetate

258

THE CAROTENOID GROUP

7

functions (M-6o), etc.; most carotenoids give prominent M-Io6 peaks (loss of xylene from the polyene chain). For a review see U. Schwiaer et al., Chimia, 1965, x9, 294, and for applications to structure determination see those publications by the groups of B. C. L. Weedon, A. Jensen and S. L. Jensen from late 1964 onwards dealing with fucoxanthin, foliaxanthin, and flexixanthin.

(c) Chemical proper6es (0 Pyrolysis Kuhn and A. Winterstein (Per., I933, 66, 429, 1733) found that on dry distillation in vacuo, lycopene, /5-carotene, and several other carotenoids gave oils from which small amounts of toluene, m-xylene, and 2,6-dimethylnaphthalene could be isolated, the latter compound arising from the central portion of the polyene chain:

.~

~

M

e

2.6- Dimethy[naphthatene Recent investigations using modern methods of separation (gas-liquidand thin-layer-chromatography) have shown that the oil obtained on vacuum pyrolysis (3oo~ of/~-carotene contains, in addition to small amounts of the above three hydrocarbons, ionene

lonene

(major product: 34% based on the /5-carotene), the 3,4-dehydro derivative of ionene, and 2,2,6-trimethyl-I-m-tolylcyclohexane. Ionene is also the major product from the mild pyrolysis of/~-carotene (in benzene at 188 ~ (F. S. Edmunds and R. A. W. Johnstone, J. chem. Soc., 1965, 2892; W. C. Day and J. G. Erdman, Science, 1963, 141, 808; cf. also I. Mader, ibid., 1964, I44, 533).

(ii) Isomerisation The isolated double bond in the ~-ionone ring of a carotenoid can be isomerised into conjugation with the polyene chain by prolonged heating

4

CHEMICAL

259

PROPERTIES

in benzene/sodium ethoxide at IOO~ a-carotene, ~-carotene, and lutein yield small quantities of /~-carotene, ~,-carotene and zeaxanthin, respectively (Karrer and E. Jucker, Helv., 1947, 30, 266; T. E. Kargl and F. W. Quackenbush, Arch. Biochem. Biophys., 196o, 88, 59):

(iii) Catalytic hydrogenation Catalytic hydrogenation over platinum oxide at atmospheric pressure converts carotenoids into the corresponding perhydro derivatives; carbonyl groups, if present, are also reduced but only relatively slowly. (Regarding the selective reduction of the acetylenic bond in synthetic intermediates cf. p. 247).

(iv) Oxidation reactions of carotenoid hydrocarbons Reactions of carotenoids with various oxidising and dehydrogenating agents are most interesting from a structural point of view. The most important are as follows. (I) With permanganate. Treatment of/~-carotene (XXXI) in benzene with cold alkaline permanganate cleaves the polyene chain as indicated to give

/~-Carotene (x'x'x'I) ('=I )

the aldehydes ~-apo-8'-carotenal and /~-apo-12'-carotena] (cf. p. 236) (P. Karrer et al., Helv., I937, 20,682, IO2O). Similarly, a-carotene (XXXII) gives ~-apo-8'-carotenal, zeaxanthin (•215 gives apo-8'-zeaxanthinal C"~citraurin"), and lycopene (XXXIV) ~ives a mixture of aldehydes and dialdehydes corresponding to fission either at one end of the molecule or at both ends simultaneously (iden;, ibid., 1938, 21, 21I, 448; z939, 22, 69): !

(~)=CII)

~.OH (~) HO

260

THE CAROTENOID

GROUP

7

~)-(m)

Prolonged treatment gives rise to further fragmentation and aliphatic dicarboxylic acids derived from the end-groups can be isolated (p. 243). (2) With chromic acid. In contrast to permanganate, chromic acid attacks the ring double bond of fl-carotene. Thus, R. K u h n and H. Brockmann (cf. Ann., 1935 , 516, 95) carried out the transformations shown in Scheme 3, using various amounts of cold o. I N-chromic acid (cf. also p. 244):

S 0

Semi - ~ - carotenone

Dihydroxy -~ - carotene.

.

0

.

.

.

.

.

HO

.

.

.

.

0

Di h y d r o x y s e m i -/~ - c a r o t e n o n e

0 ~- Carotenone

S c h e m e 3.

A similar series of derivatives can be obtained from a-carotene (XXXII) ; the fl-ionone ring is preferentially attacked, the derivatives isolated possessing an intact a-ionone ring (Karrer et al., Helv., ~934, 17, II69). (3) With hydrogen p e r o x i d e - osm,um tetroxide in tert-butanol, fl-carotene (XXXI) yields a mixture of fl-apocarotenals including a relatively large amount (lO-3O%) of the C20 aldehyde, retinene 1 (p. 289) produced by fission of the central double bond (N. L. Wendler et al., J. Amer. chem. Soc., 195o, 72, 234; cf. J. Glover, Ann. Reports, I959, 56, 334) ; (using hydrogen peroxide alone, the yield of retinene 1 is only ~ 1% : R. F. Hunter and N. E. Williams, J. chem. Soc., 1945, 554)-Zeaxanthin (XXXlII) i eacts similarly giving 3-hydroxyretinene 1 in ~ 30 % yield (R. K. Barua and A. B. Barua, Biochem. J., 1966, ioi, 250).

4

CHEMICAL PROPERTIES

261

(4) With per-acids the ring double bond is preferentially attacked and a 5,6-epoxide is formed (see p. 293 ). (5) Enzymic oxidatwn of fl-carotene is discussed by J. Friend (Chem. and Ind., z958, 597)(6) Dehydrogenation with N-bromosuccinimide*. On treatment with 2 moles of N-bromosuccinimide (in refluxing carbon tetrachloride for 2 hrs. ; conditions are important), fl-carotene gives a mixture of five products with thcee retro compounds predominating (Zechmeister et al., J. Amer. chem. Soc., 1953, 75, 4493; 1955, 77, 55, 2567) (Scheme 4).

(,~xxv[)

(xxxvH) (see p. 306)

j

-...

CXxxvm')

(Xx xix IS c h e m e 4.

a-Carotene gives XXXVI, X X X V I I , X X X I X and 3,4-dehydro-a-carotene (Zechmeister et al., loc. cir.). fl-Carotene yields essentially the same mixture if ethanol-free chloroform is used as solvent but the use of chloroform containing 1% ethanol has a marked effect: the reaction (3 moles of N-bromosuccinimide) proceeds rapidly even at --200 and, after quenching with base, yields mainly oxygenated derivatives, although small amounts of X X X V and X X X I X are also produced (F.J. PetracekandZechmeister, ibid., I956 , 78, 1427) (Scheme 5). The ethanol can be replaced by methanol or benzyl alcohol without affecting the overall reaction. Scheme 6 summarises some of these transformations (cf. R. Entschel and Karrer, Helv., 1958, 41,402, 983). * Z e c h m e i s t e r gives a g o o d r e v i e w of w o r k c a r r i e d o u t on t h i s r e a c t i o n in F o r t s c h r . C h e m . org. N a t u r s t o f f e , 1958, 15, 31.

262

THE CAROTENOID

i

i

7

GROUP

y

0

0

Echinenone

Canthaxanthin

0

(7~E.~) O Scheme 5. (For echinenone a n d c a n t h a x a n t h i n , see pp. 314, 318).

( - - H B r ) =-

9

~/~o~,

-L

/,4

~/~_%,~[~__ ;~.~ ." %

//

//

Br"

OEt

Scheme 6.

By treating/5-carotene at --180 with 2 moles (only) of N-bromosuccinimide in chloroform containing acetic acid instead of ethanol, the following transformation can be effected in 60% yield (EnLschel and Karrer, loc. cir.) (the technical synthesis of canthaxanthin is based on this process: p. 320): "Lsozeaxarrthin diacetate~ ..... ~ O A c

4

CHEMICAL PROPERTIES

263

If only one mole of N-bromosuccinimide is used, the major product is the monoacetate (isokryptoxanthin acetate); for the similar introduction of the --SPh and t h e - - N H P h groups, cf. C. Martin and Karrer, Helv., 1959, 42, 464 . With kryptoxanthin, attack occurs mainly at C(,) (Karrer and L. Jaeger, ibid., 1963 , 46, 688)"

A

~

1mole NBS _-CHCl3-AcOH

9

--~ .....

Kryptoxanthio

~

.......

AcO

(56%)

a~tate

(For the stepwise dehydrogenation of phytoene with three double bonds in conjugation (p. 335) to bisdehydrolycopene with fifteen, see pp. 334, 280). (7) Allylic oxidation reactions. (a) With tetralin hydroperoxide /5-carotene undergoes allylic oxidation giving 4-hydroxy- and 4,4'-dihydroxy-fl-carotenes, and the corresponding ketones; minor products include dehydro-fl-carotenes formed by dehydration of the hydroxy compounds (C. Bodea et al., Ann., 1959, 627, 237):

~

-"

"ROOH =-

E~-"

ROOH ~ { ~ - -

OH

(b) With lead tetra-acetate (in acetic acid) [Bodea and E. Nicoara, Rev. Chim. (Acad. R.P.R.), 1962, 7, 79]/5-carotene reacts very rapidly at 25 o to give three main products, XL, XLI, XLII (Scheme 7)/9 - Carotene( ~ )

/

OAc"

....'~~ /

OAc

OAc

\

OAc (XLI)

Scheme7-

(XLII)

(XL)

264

THE CAROTENOID

7

GROUP

retro-Dehydro-fl-carotene (XLII) yields XLI exclusively, electrophilic attack of OAc ~ replacing radical attack at the allylic position. Generally, treatment with lead tetra-acetate also produces small quantities of 5,6epoxides (in their furanoid forms" p. 293 ). (c) With boron tr~fluoride etherate (see next paragraph). (v) Reaction with boron trifluoride etherate* Carotenoids give a deep blue complex with this reagent. Decomposition of the complex formed from a carotene with water or an alcohol leads to the insertion of an oxygen function. Thus, the retro-dehydro-fl-carotene (XLII)boron trifluoride complex yields 4-hydroxy- and 4-methoxy-fl-carotene with water and methanol, respectively; fl-carotene (XXXI) (which is simultaneously dehydrogenated by the boron trifluoride) yields the same products (Zechmeister et al., J. Amer. chem. Soc., z953, 75, 4495; 1956, 78, 3188). ~-Carotene (XXXlI) similarly yields (20 to 40 %) 4-hydroxy-and 4-methoxy~-carotene (the a-ionone ring being unaffected) and lycopene reacts as shown (W. V. Bush and Zechmeis~er, ibid., 1958, 80, 2991). This reaction is always accompanied by considerable trans-cis isomerisation (Scheme 8).

OR (Xl.ltl)

BF"3 : ROll

OR

Lycopene

S c h e m e 8.

(vi) Reactions of carotenoids containing allylic hydroxyl (or alkoxyl) groups (I) Addition of a few drops of a solution of dry hydrogen chloride in ethanolfree chloroform to a chloroform solution of the carotenoid results in a visible deepening of the colour of the solution due to a spectral shift to longer wavelengths [see Karrer and E. Leumann, Helv., 1951, :34, 445 (i);Zechmeister et al., J. Amer. chem. Soc., 1953, 75, 4495 (ii); 1956, 78, 1427 (iii) ; cf. also ibid., 1958, 80, 29911 (Scheme 9). * F o r e a r l i e r w o r k on t h i s r e a c t i o n see Zechmeister, F o r t s c h r . C h e m . org. N a t u r s t o f f e , 1958, 15, 31.

4

CHEMICAL

265

PROPERTIES

~BF3;ROH OR

(ii)

(R = H o r alkyl )

(iii) OH

0

0

S c h e m e 9.

The acid-catalysed dehydration of allylic hydroxyl groups is frequently encountered in synthetic procedures (see pp. 247, 269 , 285). (2) Addition of a drop of the chloroform-hydrogen chloride reagent to a methanol solution of a carotenoid with allylic hydroxyl groups brings about their methylation (with a consequent marked effect on polarity); the reverse reaction can be carried out by replacing the methanol with aqueous acetone (Zechmeister et al., J. Amer. chem. Soc., 1956, 78, 1427; I958, 80, 299I; E. C. Grob and R. P. Pflugshaupt, Helv., 1962, 45, 1592) :

[~ -OH

.,H|

~--

H|

(3) Oxidation to the corresponding ketone, which causes a characteristic spectral shift of ~ 25 m y (i) or ~ 5 m y (ii), can be achieved"

(i)HO~---

O~F~--(ii)

R

R OH

0

Manganese dioxide effects reaction (i) in high yield in the vitamin A series (R = H; p. 289) but fails apparently if " R " is a bulky group. However, p-chloranil (tetrachloro-I,4-benzoquinone) is effective even when " R " is bulky, but fails when " R " contains a 7-hydroxyl group; 2,3-dichloro-5,6dicyano-I,4-benzoquinone has also been used, in reaction (ii) (Weedon et al., J. chem. Soc., 1958, 3972; 1961, 4oi9; Acta Chem. Scand., 1966, 20, 1195 ).

266

THE CAROTENOID

GROUP

7

In addition S. L. Jensen (ibid., 1965, 19, 1166) has found that the allylic hydroxyl groups of 4,4'-dihydroxy-fl-carotene can be efficiently oxidised to carbonyts by treating with p-chloranil in benzene-ethanol solution containing a trace of iodine as catalyst and irradiating the mixture with visible (preferably sodium-vapour) light; unfortunately, extensive trans --~ cis isomerisation occurs simultaneously. Silver oxide has also been used (cf. p. 315), and gaseous oxygen is effective with certain compounds (Bush and Zechmeister, J. Amer. chem. Soc., 1958, 80, 2991). The above conversion can be reversed by treating the polyene ketone with sodium borohydride in cold ethanol (A. L. Curl, J. agric. Food Chem., 1962, xo, 504; Weedon et al., loc. cit.) :

HO~__

~'P-Chtor-aniletc. ., ~ NaBH4

~

__

R

(vii) Reaction with bases (for acids cf. page 25o). Most carotenoids are fairly stable to base; exceptions are: (a) those containing a conjugated-ketone grouping, which undergo a retro-aldol cleavage ~- to the carbonyl group (pp. 322, 325); (b) fucoxanthin, (p. 329); (c) those with an astaxanthin-type end-group (pp. 320, 332).

5. Carotenoid hydrocarbons* Lycopene is familiar as the main red colouring matter of the tomato (Lycopersicum esculentum) from which it was first isolated in 1875. The molecular formula (C40Hse) was established in 191o by R. Willstdtter and H. H. Escher (Z. physiol. Chem., I9IO, 64, 47). By application of the technique outlined on p. 243, P. Karrer and his collaborators (Helv., 1928, zI, 751; 193o, z3, lO84; 1931, x4, 435) showed that lycopene contains two isopropylidene groups, thirteen double bonds, six side-chain methyls, and is acyclic. Perhydrolycopene was found to be identical with the product obtained by treating dihydrophytyl bromide with potassium (Karrer et al., ibid., 1928, zz, 12Ol) thus establishing the nature of the carbon skeleton. From these results, lycopene was formulated as III (cf. footnote, p. 232) (Karrer et at., I93O, 1931, loc. cir.):

(m')

Lycopene * The trans--~ cis isomerisation (cf. p. 250 ) b e h a v i o u r of m a n y of the carotenoids described here has been s u m m a r i s e d b y L. Zechmeister (Fortschr. Chem. org. Naturstoffe, I96O, 18, 223). M.p.'s quoted were generally d e t e r m i n e d in e v a c u a t e d capillaries. Where ~maxvalues are quoted, the m o s t intense p e a k is denoted b y bold t y p e and a shoulder or inflexion b y brackets.

HYDROCARBONS

5

267

This structure was confirmed b y R. K u h n and C. Grundmann (Ber., 1932, 65, 898, 188o) who showed t h a t stepwise oxidation with chromic acid gave methylh e p t e n o n e ( M e , C = C H - C H , . C H , . C O . M e ) and bixindial ~the structure of which followed from its conversion into the corresponding (known) diacid norbixin (p. 327) t h r o u g h the sequence: - - C H - - N O H - - + - - C ~ N - + - - C O , H ] so identifying the position of the chromophore. o Bixindial

Lycopene is now known to occur widely in nature, particularly in fruits and berries. Physical properties: Red needles from petrol, m.p. 173 o (Karrer and R. Widmer, Helv., 1928, zz, 75I), 172-173 o (O. Islet et al., ibid., 1956, 39, 463); ~-max. 505, 472, 446 (in petrol); 548, 507, 477 m/z (in carbon disulphide); for I.R. and P.M.R. spectra see Isler et al., loc. cir., and B. C. L. Weedon et al., J. chem. Soc., 1960, 287o. Syntheses*: (i) B y Karrer et al., (Helv., 195o, 33, I349), s t a r t i n g from the acyclic analogue (w-ionone) of fl-ionone and then following the same reaction sequence used for the synthesis of fl-carotene (see p. 268). The overall yield was, as for the fl-carotene synthesis, very low b u t was later improved somewhat (ibid., 1953, 36, 828). (ii) B y Isler et al. (loc. cir.), s t a r t i n g from linalool (XLIV) which was converted into geranyl bromide (XLV) and thence to the phosphorane XLVI; this coupled (7o%) with the C9.0 dial, crocetindial (XXlV) (p. 248) to give lycopene:

~OH (XLIV)

FBr3

~ (XLV)

CH2Br PPh3;PhLI

~

CH---pph3

(X'X'IV)

]II

(XLVI)

Natural polycis-lycopenes ; prolycopene. - About t w e n t y polycis-lycopenes (cf. p. 25 i) have so far been isolated from n a t u r a l sources including rose hips, certain strains of tomato, and swede roots; for a list, cf. L. Zechmeister, Fortschr. Chem. org. Naturstoffe, I96O, 18, 289. Frequently, several different polycis-lycopenes are found together in one plant. T h e y all have degraded visible absorption spectra, which are d r a m a t i c a l l y influenced b y iodine (see p. 255); a few have been obtained crystalline, so d e m o n s t r a t i n g their (relative) stability to heat. Zechmeister uses the term "'prolycopene" to designate one particular polycislycopene (m.p. I I I ~ isolated from a variety of t o m a t o (J. Amer. chem. Soc., 1942, 64, IO75); some authors use the terms prolycopenes I, I I . . . . etc. to designate various polycis-lycopenes. * In this and subsequent sections the earlier syntheses of Karrer's group are included for their historical interest but they are now of little preparative value having been superseded by those of Isler and others.

268

THE CAROTENOID GROUP

7

fl-Carotene. In 1831 Wackenroder isolated a crystalline colouring m a t t e r from carrots and n a m e d it carotene. In 19o7-19i o, Willstdtter et al. (Ann., 19o 7, 355, I; Z. physiol. Chem., I9IO, 64, 47) showed t h a t carotene had the formula C40H56, and carried out some preliminary chemical investigations (reaction with iodine, oxygen, etc.). Catalytic hydrogenation (to give C40H78) showed t h a t carotene contained eleven double bonds and two alicyclic rings (Zechmeister et al., Bet., 1928, 6I, 566). Karrer and his collaborators (Helv., 1929, I2, 1142; I93O, 13, lO84) demonstrated t h a t carotene contained six side-chain methyl groups and t h a t on oxidation with p e r m a n g a n a t e and on ozonisation it gave products (cf. pp. 243-244 ) consistent with the presence of a fl-ionone ring. Meanwhile, w h a t was apparently the same compound was discovered in various other plants (usually in their leaves). In 1931, Karrer et al. (ibid., 1931, I4, 614) and R. K u h n and E. Lederer (Ber., 1931 , 64, 1349) separately discovered t h a t the "carotene" obtained (as above) from various sources by simple extraction and crystallisation contained small and variable amounts ( ~ lO%) of an optically active isomer which could be removed by fractional crystallisation (when it became concentrated in the mother liquors) or, better, by c h r o m a t o g r a p h y (whereupon it was eluted more quickly t h a n the major constituent). This isomer ([~X]Cd -~- 380 ~ was named a-carotene, the major constituent ([a]Cd ~176being named fl-carotene. Repetition of the ozonisation experiment using pure fl-carotene and a comparison of the yield of geronic acid obtained with t h a t obtained from fl-ionone, indicated t h a t fl-carotene contained two fl-ionone rings. These results led Karrer et al. (Helv., 193o, 13, lO84; 1931, 14, lO33) to propose the now-accepted structure for the colouring m a t t e r (I):

-

Carotene

(I)

Confirmation of this structure was soon provided by K u h n (Ber., 1933, 66, 429; cf. Ann., 1935, 516, 95) and others, and more recently by total synthesis. fl-Carotene is one of the most widespread carotenoids in nature, and is the most a b u n d a n t carotene (cf. p. 237 et seq.). Regarding its vitamin A activity, cf. p. 283. Physical properties. Dark violet prisms from benzene-methanol, red plates from petrol; m.p. 183 o (corr.) (cf. K u h n and H. Brockmann, Ber., 1933, 66, 4o7), 18o o (uncorr., Islet et al., Helv., 1956, 39, 249); 2max. 481, 453 (hexane); 512, 484 m/~ (carbon disulphide); for visible (various solvents) and I.R. absorption spectra cf. Islet, loc. cit. ; for P.M.R. data, cf. Weedon et al., loc. cir. Syntheses (see footnote p. 267). (i) By Karrer and C. H. Eugster (Helv., 195 o, 33, II72 ), this constituting the first total synthesis of a carotenoid. The reaction sequence (Scheme IO) started from fl-ionone (XLVII) and utilised the Cs-diketone (XLVIII) described on p. 246.

5

269

HYDROCARBONS

The yield in the two-stage conversion of the C40 tetraol (L) into fl-carotene was only 3 To.

~

O

Br'CHEC=CHZn -- ~

(XLVII)

(XLIX)

2(XLIX) + 2 EtMgBr +

O.~~..~,/~~O

I

(XLVllI)

(L) I 89

BaSO4~He(- H20)

|

el) Scheme i o. (ii) Only shortly after Karrer's publication, H. H. Inhogen and his co-workers described two further syntheses of fl-carotene. In the first (Ann., 195 o, 569, 237) the same starting materials (XLIX and XLVIII) were used as were used by K a r r e r b u t the order in which the transformations were effected differed"

(XLIX) --H2% ~

PhLi;(XLVIII)

/

H2/Pd-quinoline; H|

The overall yield was, again, very low. Inho~en assumed t h a t the C~6-acetylenic h y d r o c a r b o n and the C40-diol he prepared both had end groups of the " n o r m a l " (fl-carotene) t y p e b u t a reinvestigation by K. Eiter et al. (Ann., 1965, 684, 14) revealed t h a t t h e y were in fact the isomeric retro-compounds, as shown; E i t e r also showed t h a t if the genuine " n o r m a l " C~6-hydrocarbon is p u t through Inhoffen's reaction sequence (slightly modified) fl-carotene is obtained in good overall yield. Inho!]en's second route (ibid., I95 o, 570, 54) also provided the first total synthesis of a cis-carotenoid; it was based on the fl-C14-aldehyde (XlX) used earlier in the synthesis of vitamin A (p. 285), which was converted (see below)

270

THE

CAROTENOID

GROUP

7

into the fl-C19-aldehyde (XX)" two of these Cl9 units were then linked with acetylenedimagnesium bromide to give a C,0 diol (LI) (Scheme II).

(~)

(~q~)

j / / / ~ r M g C ~CMgBr OH (LI)

I H| - H20 ); H2/Pd - quinol ine Central - cis -~ - carotene (I) Scheme i I.

The overall yield was low primarily because of the low yield (6%) obtained in the dehydration step (effected with toluene-p-sulphonic acid in hot toluene). Inhoffen converted the fl-C14-aldehyde (XlX) into the fl-Cl,-aldehyde (XX) as follows: OMe

Me'CO'CH2OMe LiC------CH

~

O

Me

CI:~, PhLi =-

LV~

(~

OH

OH H@/EtOH; H2/Pd - quinoLine

~ ~ , ~ ~ ~ ~ O M e

'c.

o.

H@

~ - -

(XZ3

(iii) In 1956, Isler et al. (Helv., 1956, 39, 249) described the first high-yield synthesis of r-carotene. This was based on Inhoffen's second route (above) b u t the overall yield was greatly improved. Thus the Cl(aldehyde (XIX) was convetted into the Ca,-aldehyde (XX) by chain extension using ethyl vinyl ether followed by ethyl propenyl ether, as on p. 246, and the yield for the dehydration step was increased to ~ 6o% by using hydrogen bromide in methylene chloride a t - - 3 o~ r-Carotene is now produced on an industrial scale using this route. r-Carotene has also been prepared from the cyclic analogue of geranyl bromide using the Wittig reaction as in the synthesis of lycopene, p. 267 (Isler et al., ibid., I956, 39, 463); by a Wittig reaction between fl-ionylideneacetaldehyde and phosphorane L I I (J. D. Surmatis and A. O/her, J. org. Chem., 1961, 26, 1171):

5

271

HYDROCARBONS

2~

+ Ph3P~~,,~~~~pph3

(I)

(irr) and from vitamin A (synthetic, p. 285) by the oxidative coupling of the derived phosphorane (H. J. Bestmann and O. Kratzer, Angew. Chem., 1961, 73, 757)"

2 ~

PPh3

02

(I)

(For other syntheses, cf. N. A. Milas et al., J. Amer. chem. Soc., I95 o, 72, 4844; Inhol~en et al., Ann., 1951, 573, I; Islet et al., ibid., 1957, 603, 129; Helv., 1957, 4o, 1256; 1961, 44, 985; H. Pommer, Angew. Chem., I96O, 72, 911 ; C. D. Robeson et al., J. org. Chem., 1964, 29, 187; Eiter et al., loc. cit.). " I s o c a r o t e n e " . - This hydrocarbon, formed by treatment of an iodine addition product of r-carotene with acetone (Kuhn and Lederer, Bet., 1932, 65, 637), is now known to be retro-dehydro-fl-carotene (XXXVI, p. 261) (Islet et al., Helv., 1956 , 39, 454). a-Carotene. As already mentioned (p. 268), a-carotene was first isolated from crude samples of r-carotene; it is conveniently obtained from carrot carotene (which contains ~ 85% of the fl-,and 15% of the a-isomer) by chromatography (cf. Karrer and O. Walker, ibid., 1933, 16, 641 ). Its structure was elucidated by Karrer, R. M o r / a n d Walker (ibid., 1933, 16, 975) ; the formation of both isogeronic and geronic acids on ozonisation proved the presence of rings of both the a- and fl-ionone types. The formula L I I I is consistent with the compound's optical activity and vitamin A activity ( ~ 50% of that of r-carotene: cf. H. J. Deuel et al., Arch. Biochem., 1945, 6, 157):

G-Carotene(LIII)~=II) a-Carotene is widespread in nature, frequently accompanying the r-isomer, although usually present in smaller amounts. Physical properties: Violet prisms from benzene-methanol or petrol; m.p. 187-1880 (corr.), [a]Cd +3850 (Karrer and Walker, loc. cit.); ~max. 474, 446, 422 (hexane) ; 511,479, 45 ~ m/~ (carbon disulphide). Syntheses: (i) Karrer et al. carried out a mixed condensation between the acetylenic carbinol X L I X used in their r-carotene synthesis and the analogous compound LIV derived from a-ionone with the Cs-diketone (cf. p. 269). (+)-aIonone gave (+)-a-carotene (m.p. 159-16o ~ (Helv., 195o, 33, 1952; 1955, 38, 61o) and (--)-a-ionone gave the (--)-a-carotene (m.p. 178~ [a]Cd--4oo~ which

272

7

THE CAROTENOID GROUP

does not occur in nature, (ibid., 1957, 40, 1676), b o t h reactions proceeding in low yield.

(LIV)

(XLIX)

(ii) Inho~en, U. Schwieter and G. Raspd (Ann., 1954, 588, 117) prepared the ~-ionone analogue of the ~-Cz4 aldehyde ( X l X , p. 269) and converted it (in 4 steps) into the ~-Czg-aldehyde (LV). The C2t unit (LVI) was prepared from the /~-Cxg-aldehyde (XX, p. 270 ) and lithium acetylide. (:k)-o~-Carotene (m.p. 161I63 ~ corr.) was obtained as follows:

i

(LVI) MeLi

~

H|

partial redr~ et~

(+-)-(LIII) (iii) Isler et al. (Helv., 1961, 44, 985) used the W i t t i g reaction to couple 15,15'-

dehydro-fl-apo-I2'-carotenal (LVII) with a phosphorane (LVIII) derived from a-ionone (cf. p. 274; from L X l a + base):

~,~CH-'-'PPh 3 (LVIII)

(LVII) (= XXIII; p. 247)

7-Carotene. I n 1933, K u h n and Brockmann (Ber., 1933, 66, 407) showed t h a t b y careful c h r o m a t o g r a p h y a third isomer could be isolated from carrot " c a r o t e n e " . This appeared just above/~-carotene and was present to the e x t e n t of only ~ o. 1%; it was n a m e d 7ocarotene. I t showed v i t a m i n A a c t i v i t y b u t gave acetone on ozonisation. These and other observations led to structure L I X (Kuhn and Brockmann, loc. cit.) :

~ - Carotene (LIX)

5

HYDROCARBONS

273

r-Carotene is considerably less abundant in nature than fl- or a-carotene; it has about 45% of the vitamin A activity of fl-carotene (Deuel et al., Arch. Biochem., I949, 23, 242). Physical properties: Dark red prisms from benzene-methanol; quoted m.p.'s for analytically pure samples vary from I3I to 1780 (cf. Zechmeister et al., Arch. Biochem., I95 o, 26, 358); K u h n and Brockmann (loc. cit.) quote 178~ Isler et al. (Helv., I96I, 44, 985) were unable to raise the m.p. of natural or synthetic material above I52-I54~ ;tmax. 494, 462, 437 (petrol); 533, 496, 463 m/z (carbon disulphide); for I.R., visible, and P.M.R. spectra, cf. Islet et al. (loc. cir.). Syntheses: (i) C. F. Garbers, C. H. Eugster and Karrer (Helv., I953, 36, I783) obtained r-carotene in low yield by carrying out a mixed condensation on the same lines as for a-carotene. (ii) Isler et al. (loc. cir.) carried out a Wittig reaction between fl-apo-8'-carotenal (cf. C30: p. 247 ) ( L X ) a n d the phosphorane XLVI derived from geranyl bromide (p. 267) and obtained r-carotene in good yield:

+ ph3p=~"~ (XLVI)

(LIX)

Further syntheses include: (a) (Isler, 1961, loc. cit.) a Wittig coupling of the fl-C=5-carotenal (LVII) and the acyclic analogue (derived from ~-ionone) of compound LVIII [cf. synthesis (iii) under a-carotene], and (b)(Ri~egg et al., J. chem. Soc., 1965, 2o19) a two-stage Wittig coupling (C10 dial--+ C25 monoaldehyde ---+ C40 hydrocarbon). Pro-r-carotene. This polycis form of r-carotene has been found in various natural sources and is crystalline (m.p. 135~ it has the properties generally associated with a polycis carotene (cf. under polycis-lycopenes, p. 267: Zechmeister fists its natural occurrence); the berries of Pyracantha angusti/olia provide a practical source (Zechmeister and W. A. Schroeder, J. biol. Chem., 1942, x44, 315; J. Amer. chem. Soc., 1942, 64, 1173). b-Carotene. This carotene normally occurs only in trace amounts in tomatoes, but by special breeding a "high b-carotene" strain can be obtained (cf. J. w. Porter and R. E. Lincoln, Arch. Biochem., 195o, 27, 39o). Structure L X I was proposed for b-carotene following extensive degradative work (cf. T. E. Kargl and F. W. Quackenbush, Arch. Biochem. Biophys., I96O, 88, 59); its lack of vitamin A activity and its observed optical activity ([a]Cd +317 ~ (Porter and M. M. Murphey, ibid., 1951, 32, 21) are both in accordance with this:

6-Carotene(LXI)

274

THE CAROTENOID GROUP

7

6-Carotene was first detected in the tropical fruit Gonocaryum pyri[orme (A. Winterstein, Z. physiol. Chem., 1933, 219, 249) and also occurs in carrots (H. H. Strain, J. biol. Chem., 1939, 127, 191). Physical properties. Red needles, m.p. 14o. 50 (Porter and Murphey, loc. cir.) ; 2max. 488, 456, 43o m/~ (hexane); [~], see above. Syntheses: (three routes: R. Riiegg et al., J. chem. Soc., 1965, 2oi9) :

(I- Ionone

(i) LiC ~ C H (i!) H2/Lindlar" c:~'-~-

Ph3P. HBr '

-~v

~v

/CH2PPh3 BrO

(LXI=)

A similar series of reactions from w-ionone gave (45%) the analogous acyclic Wittig salt. Each of these Wittig salts was then made to react under carefully controlled conditions with I mole of 2,7-dimethyloctatrienedial (from partial reduction and acid treatment of XXVI, p. 248). The resulting cyclic and acyclic C=5 polyene aldehydes (45%} were isolated and then linked separately (6o-8o%; Wittig reaction) with the acyclic and the cyclic Wittig salts to give (=t=)-d}carotene from each. A third synthesis used a Wittig reaction to link a C=0 unit built up from =-ionone with the acyclic analogue of retinene. e-Carotene was first isolated from the diatom Navicula torquatum by Strain and W. M. Manning (J. Amer. chem. Soc., 1943, 65, 2258}; its polarity and absorption spectrum (/~rnax. 47o, 44x, 417 m/~, in ethanol} were indicative of a hydrocarbon containing a nonaene chromophore, e-Carotene has only rarely been found elsewhere (cf. D. ]. Chapman and F. T. Haxo, Plant and Cell Physiol., 1963, 4, 57; and also H. H. Trombly and ]. W. Porter, Arch. Biochem. Biophys., 1953, 43, 443, who detected a compound with similar properties in a tomato mutant}. Chapman and Haxo (loc. cir.) have since shown t h a t e-carotene is identical* with a C40 nonaene hydrocarbon synthesised by Karrer et al. and named by them "ex-carotene" [structure below; obtained as for fl-carotene (p. 268) but starting from ~-ionone: Helv., 195o, 33, 1433; I958, 4 x, 32]. Much improved syntheses of e-carotene (as a mixture of racemic and meso forms*} have recently been reported - (a) based on the coupling (Wittig reaction) of two moles of Cx5 end group with 2,7-dimethyloctatrienedial (from XXVI, p. 248) and (b) on the Wittig coupling of two C=o units (C=0 aldehyde + corresponding phosphorane) (Riiegg, loc. cir.).

s

-

Carotene

Thus, all six hydrocarbons derivable from a central nonaene chain and =-, fl- and ~-ionone end groups have been found in nature: this is of biogenetic interest. * The configuration of natural e-carotene at Ce and Ca,, is unknown.

5

275

HYDROCARBONS

~-Carotene. In 1939, Strain detected a polyene hydrocarbon in carrots which absorbed at considerably shorter wavelengths than other carotenoids known at that time and which subsequently he named ~-(zeta)carotene (Strain et al., J. biol. Chem., 1939, I27, 191; J. Amer. chem. Soc., 1943, 55, 2258)-Since then ~-carotene has been found widely in nature (cf. p. 334 et seq.); carrot oil (a commercial extract prepared from dehydrated carrots) is a convenient source. It is of considerable biogenetic interest as it appears to be the natural precursor of all, or most, of the other carotenes (cf. p. 339, 336). The elucidation of its structure has been hampered by its unusual sensitivity to atmospheric oxidation. H. A. Nash, Quackenbush and Porter (ibid., 1948, 7o, 3613) showed that it was a C40, isoprenoid (C-methyl determination) hydrocarbon with a heptaene chromophore a n d probably acyclic (isopropylidene-group determination); they tentatively assigned it an octahydrolycopene structure. More extensive degradative work and a P.M.R. study eliminated this suggestion and reduced the number of possible structures to two, namely, L X I I and an analogous compound with the chromophore at C(5)-C(~s'). (Rabourn and Quackenbush, unpubl. ; Weedon et al., Proc. chem. Soc., 1961, 261; J. chem. Soc. [C], 1966, 2154); of these, that shown was proved to be the correct structure by total synthesis of both compounds and comparison of each with the natural material (P. T. Siddons and Weedon : cf. Weedon et al., loc. cit.) :

Physical properties: m.p. 38-42~ 2max 425, 4ox, 380 m/~ (hexane) (Weedon et al.). Synthesis: (Siddons and Weedon, loc. cir.): CH2PPh 3 Br

PBr3 ; Pl::)h3~_Nerotidot (LX,,,) (LXIIIa)

~ B u L i / ~ ~ (d. XXVl p. 2 4 8 ~ 0 (LXII}

Cx-Carotene, m.p. 72-74 ~ and of unknown structure, accompanies C-carotene in corn but is far less labile (E. N. Petzold, Diss. Abs., 1959, 20, 62). ~7-Carotene. In 1952, T. W. Goodwin (Biochem. J., 1952, St, 458) detected a pale yellow carotenoid in the ripe berries of Lonicera japonica (honeysuckle) and named it r/-(eta)-carotene. A comparison of its spectral (2max. 425, 399 m/~, in petrol) and chromatographic properties with those of ~-carotene suggested t h a t it may be a cyclic analogue of ~-carotene. ~7-Carotene has also been detected in swede roots (A. E. Joyce, J. Sci. Food Agr., 1959, xo, 342). 0-Carotene, a carotenoid of unknown structure (itmax. 421, 397, 375 m/~, in hexane), occurs in the fungus Neurospora crassa (Haxo, Fortschr. Chem. org.

276

THE C A R O T E N O I D GROUP

7

Naturstoffe, 1955, 12, 175 ) and possibly elsewhere (cf. T. O. M. Nakayama, Arch. Biochem. Biophys., 1958, 75, 356). Neurosporene. In 1949, Haxo (Arch. Biochem., 1949, 2o, 4oo) isolated a C40 hydrocarbon with a nonaene chromophore from the fungus Neurospora crassa and named it neurosporene. Neurosporene is otherwise of rather limited occurrence; it has most frequently been found in organisms (often fungi) prevented from producing their normal carotenoids by artificial interference [by growing the organisms in the presence of diphenylamine (cf. p. 334) or by changing their genetic make-up by X-ray or ultra-violet irradiation (Claes, Z. Naturforsch., 1956, xxb, 26o; Nakayama, Arch. Biochem. Biophys., 1958, 75, 352, 356)]. Until recently neurosporene was thought to be 5,6,5',6'-tetrahydrolycopene (and is frequently called "tetrahydrolycopene" in the literature) mainly by analogy with the structure which was first assigned to ~-carotene (and now known to be incorrect: cf. p. 275 ). The correct structure LXIV followed from its P.M.R spectrum (which eliminated the tetrahydrolycopene structure and showed peaks for two "end-of-chain" and three "in-chain" methyls, two pairs of isopropylidene methyls, and one methyl on an isolated trans-double bond: cf. p. 257), and was confirmed by total synthesis (Weedon et al., Proc. chem. Soc., 1961, 261; J. chem. Soc. [C], 1966, 2154):

Neurosporene (LXIV) ( .7, 8 - dihydrotycopene )

Physical properties: m.p. 1240 (naxo, loc. cit.); )-max. 470, 440, 416 m/~ (hexane). Synthesis: Siddons and Weedon (cf. Weedon et al., loc. cir.) synthesised neurosporene starting from ~-ioaone and nerolidol (LXlII). These were converted (7 steps) into the polyene aldehyde L X V I and (6 steps) the phosphonium bromide LXV, respectively; in each reaction sequence, chain extension was effected as follows (Horner reaction: cf. Pommer, Angew. Chem., 196o, 72, 911): o R

+

II

(EtO) 2 P. CH

C02Me

NaOMe

--~

C02Me

R

The two Cz0 units were linked by the Wittig reaction:

o + P%P.CH2 ar e (LXVI)

I

I

/ \

BuLi__~_ (LXIV)

(LXV)

Proneurosporene (sometimes reported as "protetrahydrolycopene": cf. above). This non-crystalline polycis-lycopene occurs in certain strains of tomatoes (Porter et al., Arch. Biochem., I95 o, 27, 390; Arch. Biochem. Biophys., 1953, 43, 443) and in the berries of Pyracantha angustilolia (E. F. Magoon and Zechmeister, ibid., 1957, 68, 263).

5

HYDROCARBONS

277

~-Zeacarotene and ~-zeacarotene. Over the period 194o-1953 several workers reported t h a t even after repeated c h r o m a t o g r a p h y one of the carotenes isolable from corn showed an unusually complex absorption spectrum (~max. ~ 450, 425, 4oo, 380 m/,, in hexane) (cf. E. C. Callison et al., J. lXlutrit., 1953, 5 o, 92; C. S. Boru~ et al., Ind. Eng. Chem., 1944, 35, 344; etc.). In 1959, Petzold, Quackenbush and M. McQuistan (Arch. Biochem. Biophys., 1959, 82, 117) found t h a t b y using more active adsorbents this " c a r o t e n e " could be resolved into three major components - ~-carotene and two new colouring matters, which were n a m e d ~-zeacarotene and fl-zeacarotene (~.max. 449, 421, 398 and 454, 427, 4o7 m/~ respectively, in hexane). T r e a t m e n t of these two pigments with N-bromosuccinimide converted t h e m into Q-carotene (LXI) and ~,-carotene (LIX), respectively, and so the following structures were proposed:

(LXVII)

( LXVIII )

( (:Z-Zeacarotene ? )

/ ~ - Zeacarotene (proba~'Cy)

In accordance with these proposals ~-zeacarotene b u t not ~-zeacarotene was found to have v i t a m i n A activity. In 1961, Islet et al. (Helv., 1961, 44, 994) synthesised c o m p o u n d L X V I I I [via a W i t t i g reaction between a C=5 aldehyde ( X X I I I , p. 247 ) and farnesyl bromide]. The synthetic material (a solid, m.p. 96-97 ~ also gave 7-carotene on t r e a t m e n t with N-bromosuccinimide, and had the same absorption curve and chromatographic properties as the n a t u r a l (as y e t uncrystallised) fl-zeacarotene (Isler, 1961, loc. cir.; K. L. Simpson and Goodwin, Phytochem., 1965, 4, I93). Isorenieratene, renieratene and renierapurpurin. These three isomeric (C40H48) hydrocarbons were isolated (relative yields, I o: 20: I) from a sea sponge (Reniera japonica) by M. Yamaguchi (Bull. chem. Soc. Japan, 1957, 3o, i i i , 979; 1958, 3x, 51,739). Oxidation of either isorenieratene or renieratene with chromic acid gave crocetindial (C=0) (XXIV), and on microhydrogenation b o t h colouring m a t t e r s absorbed 15 moles of hydrogen. Mild oxidation with p e r m a n g a n a t e gave a C30 polyene aldehyde ("isorenieral") and 2,3,6-trimethylbenzaldehyde from isorenieratene and a mixture of two C30 aldehydes (isorenieral and "renieral") and 2,3,6- and 2,3,4-trimethylbenzaldehydes from renieratene. The structures

Isorenieratene

(LXIX)

278

THE CAROTENOID GROUP

7

I\ Renieratene

(LXX)

proposed (LXIX and LXX) were supported by other evidence; all oxidation products arose by fission of the double bonds next to the aromatic rings. Too little renierapurpurin was available for degradative work but it apparently bore a close resemblance to the other two and Yamaguchi suggested that it had structure L X X I . This was subsequently confirmed by synthesis.

Renierapurpurin (LXXI)

Physical properties: Isorenieratene, purple needles from chloroform-ethanol, m.p. 199~

;tt,ax. 520, 484, (452) m#* (carbon disulphide); see also under

"leprotene" (below). Renieratene, purple needles, m.p. 185~ ;tmax. 532, 496, (463) m#* (carbon disulphide). Renierapurpurin, purple plates, m.p. 230o; ~.max. '~ 538, 504, (477) m#* (carbon disulphide) (Yamaguchi, loc. cir.); for P.M.R. and I.R. data, cf. Weedon and co-workers, J. chem. Soc., 1963, 5637, and Yamaguchi, loc. cir. None of the three colouring matters shows vitamin A activity.

Syntheses: (i) Yamaguchi (Bull. chem. Soc. Japan, 1959, 32, 1171) and later M. C. Khosla and Karrer (Helv., 196o, 43, 453) synthesised isorenieratene using the type of reaction sequence used in Karrer's synthesis of fl-carotene (p. 268); the Cls acetylenic carbinol was prepared as follows and then condensed with the Cs-diketone (XLVlII; p. 269): Me2CO.._

HC--=C.CH2Br._ Zn

Yamaguchi (Bull. chem. Soc. Japan, 196o, 33, 156o) prepared hydrocarbon L X X I in an analogous fashion and showed it to be identical with natural renierapurpurin. A mixed condensation of the 2,3,6- and 2,3,4-trimethylphenylC16-carbinols gave a mixture of isorenieratene, renieratene, and renierapurpurin separable by chromatography. All these syntheses proceeded in low yield. (ii) More convenient syntheses involve Wittig reactions with crocetindial (C2o: XXIV, p. 248); for example: .

CHO 3 steps (75 % over'all )

1

~CH2PPh3 Br(~

BuLi;C2odiaL

[sorenieratene ( L XIX )

* Regarding the positions of the light absorption maxima of these three colouring matters, cf. p. 254.

5

HYDROCARBONS

279

The isorenieratene separated out from the final reaction mixture in a high state of purity and in 9o% yield. Renierapurpurin (LXXI) was prepared similarly. A mixed condensation gave a mixture of the three hydrocarbons from which renieratene (42%) was isolated by chromatography (R. D. G. Cooper, J. B. Davis and Weedon, J. chem. Soc., 1963, 5637). "Leprotene" was the name given to a colouring matter [m.p. 198-2oo~ ~max. (CS,) 517, 479, 447 m/~; structure not determined] which Y. Takeda and others found in various members of the bacterial family Mycobacteriaceae (Takeda et al., Naturwiss., 1937, 25, 27; Z. physiol. Chem., i94 o, 265, 233; Goodwin and M. Jamikorn, Biochem. J., 1956, 62, 269, 275). However, a recent direct comparison (absorption spectra, mixed chromatograms, mixed m.p., etc.) of leprotene with isorenieratene (LXIX) has shown that the two colouring matters are identical (S. L. Jensen and Weedon, Naturwiss., 1964, 51, 482; Acta Chem. Scand., 1964, I8, 1562). Chlorobactene, the major carotenoid* in each of three species of green photosynthetic bacteria recently investigated by Jensen, E. Hegge and L. M. Jackman (Acta Chem. Scand., 1964, 18, I7O3), has m.p. I47-i48~ (red needles from acetonepetrol), ~max. 491, 46o, 434 m/, (petrol), and is a hydrocarbon (polarity; analysis). Its P.M.R. spectrum clearly showed the presence of both a trimethylphenyl end-group (sharp methyl bands near 9 7"75, and two protons coinciding at z 3 "o4) and a lycopene-type end-group (methyl bands at $ 8.39 and 8.33; and at $ 8.19). The substitution pattern in the aromatic ring was inferred from the absorption curve (maxima at shorter wavelength than might be e x p e c t e d - due to di-ortho substitution: cf. p. 254) and by analogy with known aryl carotenoids (see above). Jensen et al. therefore proposed the following structure; total synthesis of this compound by Weedan's group (below) and comparison of natural and synthetic samples provided confirmation.

Chtorobactene

Synthesis: using a two-stage Wittig reaction (R. A. Bonnett, A. A. Spark and Weedon, Acta Chem. Scand., 1964, I8, 1739 ) . Crocetindial (C20: XXlV, p. 248 ) was treated with one mole of the phosphorane derived from geranyl bromide (XLVI; p. 267) to give a mixture from which the desired C30 aldehyde was isolated by chromatography; this was then coupled with the phosphorane derived from 2,3,6-trimethylbenzyl bromide Ecf. synthesis (iil of isorenieratene, above]. A further aryl carotenoid,/5-isorenieratene, (one end group of the isorenieratene type and the other as in /~-carotene) has recently been detected (along with isorenieratene, chlorobactene, and /~-carotene) in certain brown photosynthetic bacteria (Jensen, ibid., 1965, I9, lO25); for its total synthesis, see Cooper, Davis and Weedon, 1963, loc. cit. * Accompanied by a hydroxylated derivative ("OH-chlorobactene", p. 3o5) in each case.

280

THE

CAROTENOID

7

GROUP

Torulene. This carotenoid was first isolated from the red yeast Torula rubra by Lederer (Compt. rend., 1933, z97, 1694). The structure remained unknown until 1961 when Isler and his collaborators (Helv., 1961, 44, 994) synthesised hydrocarbon L X X l I and showed it to be identical with a sample of torulene isolated from a micro-organism by Winterstein et al. (Bet., 196o, 93, 2951) :

Torutene (LXXII)

Physical properties: Fine red needles from benzene-methanol, m.p. 183-1840 (Isler et al., loc. cir.); 185 o (Lederer, loc. cir.); Zmax. 518, 484, 46o m/~ (petrol); for I R and visible absorption spectra see Islet et al., loc. cit. Synthesis: The phosphonium bromide derived from 7, 7-dimethylaUyl bromide was coupled with fl-apo-4'-carotenal (Cs~: p. 247) ( L X X l I I ) using the Wittig reaction (Islet et al., loc. cir.):

OH2PPhaBr@ | Me2C=CH

(LXXIII)

NaOEt ~--LXXll

Bisdehydrolycopene. A carotenoid detected in trace amounts in Valencia blood-oranges by Winterstein et al. (loc. cir.) was identified with synthetic (see below) bisdehydrolycopene by comparison of chromatographic behaviour and absorption properties:

Bisdehydrotycopene Syntheses: (i) Karrer and J. Rutschmann (Helv., 1945, 28, 793) treated lycopene with 2 moles of N-bromosuccinimide and obtained the bisdehydro-compound, Amax. 6Ol, 557, 520 (carbon disulphide), and 542, 5o4, 476 m/~ (hexane). (ii) A total synthesis of the colouring m a t t e r [m.p. 24 o~ (decomp.),).max. 568, 528, 492 (pyridine); 54 o, 5xo, 48o m/, (petrol)] has been described by J. D. Surmatis and A. O/net (J. org. Chem., 1963, 28, 2735):

Me2CO + HC~CH ~

,

OH

~I~OH

(i) H2/Lind[ar' cat_ (ii) Ph3P.HBr"

(i) Diketene (ii) Pyrolysis~ ~CH:~

e PPh3 Br.O

~ ~

C2odial.( ~ ) (NaOMe)

LiC--CH

Bisdehydrotycopene

5

281

HYDROCARBONS

Monodehydrolycopene, 3,4-dehydrolycopene, has been similarly detected in several (unspecified) microorganisms by Winterstein et al. (loc. cir.) and in certain fungi (S. L. Jensen, Phytochem., 1965, 4, 925). Synthesis. B y using only one equivalent of N-bromosuccinimide in the reaction with lycopene (above), Winterstein et al. (loc. cir.; cf. also Jensen, Acta Chem. Scand., 1963, I7, 489) obtained a mixture from which monodehydrolycopene was isolated and crystalLised; it was not obtained entirely pure (m.p. x96~ Amax. 574, 536, 5Ol (carbon disulphide); 535, 5oo, 468 m/~ (petrol). Synthetic analogues o/the carotenes. The following higher homologues have been synthesised using routes originally developed for the synthesis of fl-carotene:

) (i) 16,16"-Homo-i~-caroteneo

C42 Hse

Synthesised by Inhoffen et al. (Ann., I95I , 573, I); m.p. 19o ~ itmax. 496, 466, 445 m/z (ether); it has 20% of the vitamin A activity of fl-carotene (Deuel et al., Arch. Biochem. Biophys., 1952, 4o, 352).

o|) Oecepreno -13 - carotene, C5oH68

Synthesised by Karrer and C. H. Eugster (Helv., 1951, 34, 28) (low yield); Isler et al. (Ann., 1957, 603, 129); Surmatis and O/her (J. org. Chem., 1961, 26, 1171); it has m.p. 193-195 ~ (uncorr.), ~max. 538, 504, 477 m/~ (petrol) and is not significantly vitamin A active. (iii) The a-ionone analogue of decapreno-fl-carotene: synthesised by Karrer's group (Helv., 1951, 34, 823); has m.p. 217 ~ (corr.), ~tmax. 528, 495, 463 m/~ (petrol). (iv) Dodecapreno-fl-carotene, Ce0H80 (nineteen conjugated double bonds), was (probably) obtained synthetically by Karrer and Eugster (ibid., 1951, 34, 18o5); m.p. 19o-1 ~ Amax. 571, 531, 495 (petrol); 624, 579, 542 m/~ (carbon disulphide). (v) The following aryl analogues of fl-carotene have been synthesised :

Ar~

A

r

(a) Ar = C6H5, ;tmax. 536, 5o1, 475 m/z (carbon disulphide): Karrer et al., ibid., I952, 35, II79; Weedon et al., J. chem. Soc., 1963, 5637. (b) Ar -- fl-naphthyl (notable for its low solubility), ;tmax. 55o, 5x5, 487 m # (carbon disulphide): Karrer et al., Helv., I955, 38, I869.

282

THE C A R O T E N O I D GROUP

7

(vi) C o m p o u n d s c o n t a i n i n g a d d i t i o n a l (2,2'-dimethyl-; idem, ibid., p. 1359; 1956, 39, 686), modified (e.g. one of the gem-methyls replaced b y m e t o r - - C H 2 9 CHMeg; Surmatis et al., J. org. Chem., 1958, 23, I57), or fewer (I3,i3'-bisdesm e t h y l ; Inhol~en et al., Ann., 195o, 569, 226) alkyl s u b s t i t u e n t s as c o m p a r e d with fl-carotene h a v e also been synthesised, and their v i t a m i n A activities m ea s u r e d .

6. The vitamins A and related compounds

(a) Vitamin A 1: retinol*

Vitamin A (the suffix is usually omitted except where this leads to confusion) occurs, as such or esterified, in small quantities in dairy produce and in relatively massive quantities in some fish liver oils (notably halibut and tuna). Lack of vitamin A (or a suitable precursor, see below) produces several deficiency symptoms including arrest of growth, xerophthalmia (an eye disorder), and night blindness (cf. T. Moore, "Vitamin A", Elsevier, Amsterdam, 1957; Vitamins and Hormones, 196o, 18, 289-571). The early work on vitamin A was done on concentrates from fish liver oils, the vitamin being detected and assayed by its ultraviolet spectrum (~max. 325 m/z) and by the intense blue colour (~max. 62o m/~) produced with antimony trichloride in chloroform (the Carr-Price reaction) (cf. e.g., I. M. Iteilbron el al., Biochem. J., 1932, 26, 1178). In 1931-1933, P. Karrer, R. Morf and K. Sch@p (Helv., 1931, 14, 1431; I933, z6, 625), working with a concentrate, allotted vitamin A the now-accepted structure, (LXXIV) :

~ Vitamin

CH20H

A1 (retinol)

(LXXIV)

[A different numbering system is occasionally used in which the carbon carrying the hydroxyl is C(1). ] Vitamin A, C~oH~9OH, was first isolated pure and crystalline (m.p. 63-64 ~ b y

J. G. Baxter and C. D. Robeson (cf. J. Amer. chem. Soc., 1942, 64, 2411). The acetate, m.p. 57-580 (ibid., p. 2407) is a c o n v e n i e n t d e r i v a t i v e ; it has t h e same biological a c t i v i t y as the v i t a m i n itself b u t is m o r e r e s i s t a n t to a t m o s p h e r i c oxidation. [For o t h e r properties cf. Section (e), p. 290]. * The official (I.U.P.A.C.) present-day names for vitamin A, the corresponding aldehyde ("retinene"), and the corresponding acid ("vitamin A acid") are retinol, retinal, and retinoic acid; however, the traditional names are still widely used in the literature.

6

VITAMINS A

283

(i) T h e conversion of carotenoids into v i t a m i n A The relationship between carotenoids and v i t a m i n A was firmly established in 193o by Moore (Biochem. J., 193 O, 24, 692) who fed massive doses of " c a r o t e n e " (mainly fl-carotene) to rats and found t h a t v i t a m i n A a c c u m u l a t e d in the liver. I t is now known t h a t all carotenoids containing an u n s u b s t i t u t e d fl-ionone ring can act as precursors for v i t a m i n A in the animal body (i.e. are " v i t a m i n A active", or are " p r o v i t a m i n s A"), their activity being ~ 5 0 % of t h a t of flcarotene. (The a c t i v i t y of b o t h v i t a m i n A and of the p r o v i t a m i n s A depends on their stereochemistry, the all-trans form being more active t h a n a n y of the corresponding cis isomers: for lists of data, see S. R. A m e s et al., J. Amer. chem. Soc., 1955, 77, 4134, 4136; L. Zechmeister, Fortschr. Chem. org. Naturstoffe, 196o, I8,332).Carotenoids possessing a fl-ionone ring in which the 5,6 bond is epoxidised (e.g., fl-carotene diepoxide, a-carotene epoxide) also show high activity*, deoxygenation p r o b a b l y occurring in vivo (H. v. Euler and Karrer, Helv., 195o, 33, 1481). Other n a t u r a l carotenoids are inactive. Several entirely synthetic compounds s t r u c t u r a l l y related to v i t a m i n A show weak a c t i v i t y (E. R. H. Jones et al., J. chem. Soc., 1949, 2023; B. C. L. Weedon et al., ibid., 1958, 1855; O. Isler and P. Zeller, Vitamins and Hormones, 1957, I5, 31; H. O. H u i s m a n et al., Tetrahedron, I966, 22, 265), while the 9- and I 3 - d e s m e t h y l derivatives of v i t a m i n A itself show very little activity (idem, ibid., 1966, 22, 293); 2,2'-dimethyl-fl-carotene is very active as a p r o v i t a m i n A (cf. pp. 28I, 282 for refs. and o t h e r examples). The degree of v i t a m i n A activity is usually deduced from the growth response of v i t a m i n A-starved rats (cf. P. L. Harris et al. in "Methods of Biochemical Analysis", Vol. 4, Ed. D. Glick, Interscience, New York, 1957). The conversion fl-carotene (C,0Hss)--+ v i t a m i n A (C20H29OH) occurs, in m a m m a l s , in the intestinal wall (J. Glover, T. W. Goodzvin and R. A . Morton, Biochem. J., 1948, 43, 512; R. F. Krause and H. B. Pierce, Arch. Biochem., 1948, 19, 145; S. K . K o n and S. Y . Thompson, Brit. J. Nutrit., 1951, 5, 114), the v i t a m i n t h e n being stored in the liver (mainly as the p a l m i t a t e : cf. J. Ganguly et al., Biochem. J., 1963, 88, 531, 534)The m e c h a n i s m of the conversion has been the object of m u c h research, the two m a j o r hypotheses being (I) t h a t central fission of the fl-carotene occurs, so yielding two moles of v i t a m i n A per mole of fl-carotene and, (2), t h a t stepwise oxidative degradation occurs s t a r t i n g from one end of the fl-carotene molecule and so yielding one mole of v i t a m i n A. Biological assays, which showed t h a t one mole of fl-carotene produces the same growth effect as a p p r o x i m a t e l y one mole (only) of v i t a m i n A (T. H. Mead, S. W. F. Underhill and K. H. Coward, ibid., 1939, 33,589; E . M . Hume, Brit. J. Nutrit., 1951, 5, l~ provided strong s u p p o r t for scheme (2) b u t Glover's results (cf. Vitamins and Hormones, 196o, I8, 371) indicated t h a t this could not be the only p a t h w a y in operation. An in vitro s t u d y of the effect of a ceil-free e n z y m e p r e p a r a t i o n from rat intestine on l*C-labelled flcarotene has shown t h a t in the presence of molecular oxygen (which is essential) * N.B. Some doubt has since been cast on this report: cf. H. R. Cama et al., Biochem. J., 1966, 99, 308.

284

THE CAROTENOID

GROUP

7

the iS-carotene is rapidly converted into vitamin A aldehyde (= "retinene1": p. 2891, and in a yield of almost two moles per mole of /~-carotene consumed

(O. S. Goodman and H. S. Huang, Science, 1965, 149, 879; cf. also J. A. Olson and O. Hayaishi, Proc. Natl. Acad. Sci. U.S., 1965, 54, I364; K. Harashima, Biochim. Biophys. Acta, 1964, 9o, 2ii), so that the major pathway in this, albeit "unnatural" system must be (I). In vivo, the aldehyde so formed would (due to the presence of additional cofactors) be then quickly reduced to the vitamin itself (cf. Glover, Goodwin and Morton, Biochem. J., 1948, 43, lO9; and also Goodman and Huang, loc. cit.). (ii) Syntheses oJ vitamin A and related compounds Vilarnin A. (I) In 1937 K u h n and C. J. O. R. Morris (Ber., 1937, 70, 853) described the synthesis of a crude oil which apparently contained ~ 7"5% of vitamin A (biological assay; Carr-Price reaction). The route from/~-ionone is outlined in Scheme 12.

~

~1% O

,.~C02Et

BrCH2.CO2Et_ Zn

~NMHeMgI

-Ionone 0 R ~ ~

(LXX'V)

NH.C6H4.Me PCts;CrC12~_ R~ - - - v / % Me2C--C.CHO (base)

R Scheme

~

N.C6H4.Me

O (LXXVI).

H|

-At(OPri)3=- (LXXIV)

12.

However, later workers were unable to repeat this synthesis and some doubt was therefore cast on its validity (cf. Heilbron el al., J. chem. Soc., 1942, 727). (2) Kuhn and Morris's claim has since been substantiated by J. F. Arens and D. A. van Dorp (Rec. Trav. chim., 1948 , 67, 973; Produits Pharm., 1949, 4, 297) who synthesised the C15-aldehyde (LXXV) by the following route, showed that its properties agreed with those reported by Kuhn, and obtained genuine vitamin A aldehyde (LXXVI) from it using Kuhn's condensation reaction:

BrMgC----C.OEt~_- ~

O

E

t

H2 ;

H~

LXXV

(3

VITAMINS A

285

(3) In x946, 0. Islet et al. (Experientia, 1946, 2, 31) reported the synthesis of vitamin A methyl ether which, although not obtained crystalline, was essentially pure and showed very high activity (it was obtained crystalline later; see idem., Helv., 1949, 32, 489). In 1947 the same group described the first rational synthesis of the vitamin itself (idem, ibid., 1947, 30, I9II), using a route similar to that suggested earlier by Heilbron et al. (loc. cit.). This reaction sequence, which starts from fl-ionone and methyl vinyl ketone, is now used for the manufacture of vitamin A (cf. Isler el al., Chimia, 1961, 15, 2o8) (Scheme 13).

~

O

+

NaOMe ;OHE) =

CH2CI.C02Et

~

O -,

(/~ - C.4- a[clehyde )

0~

, EtMgBr ~_-

H|

NaC-~CH

OH CH2OH (LXXVII) (i) Partial redn.

(ii) AcCt

OH ~ C H 2 O A c

(LXXVIII)

12/hOt petrol ( -- I-~O);OH(3

att-trans Vitamin A~ (LXXIV)

S c h e m e I 3.

* A s m a l l a m o u n t of t h e trans c o m p o u n d is also p r o d u c e d (O. Islet et al., V i t a m i n s and H o r m o n e s , I96O, 18, 3Ol). F o r the partial r e d u c t i o n s t e p (i), cf. p. 247.

(4) Since 1947 many other routes to vitamin A have been devised. Several of these have been directed towards the preparation of cis isomers. Thus if the cis form (LXXVII) of the methylpentenynol used in the above synthesis is replaced by the trans form, then the I3-trans form (LXXIX) of the C~0 diol (LXXVIII) is produced. If now L X X I X and L X X V I I I are separately sub~

~

~

O

R

CH2OH

(LXXX)

(LXXIX)

(LXXVIII)

CH.dZ)H

CH2OH

(LXXXl)

286

THE CAROTENOID GROUP

7

mitted to the reaction sequence, mild acetylation, dehydration, hydrolysis, and partial hydrogenation (cf. p. 247), the (hindered*) I I - c i s - ( L X X X ) and II,I3-di-cis-(LXXXI ) isomers of vitamin A are obtained (W. Oroshnik, J. Amer. chem. Soc., 1956, 78, 2651). (5) The (unhindered*) 9-cis, I3-cis, and 9,I3-di-cis isomers have been s3rnthesised (C. D. Robeson et al., ibid., 1955, 77, 4 I l l ; M. Matsui et al., J. Vitaminol., 1958, 4, 178) by reduction of the methyl esters of the corresponding synthetic vitamin A acids (see below) with lithium aluminium hydride. (6) Vitamin A syntheses based on 2,2,6-trimethylcyclohexanone (prepared by controlled methylation of cyclohexanone) have also been recorded (J. Attenburrow et al., J. chem. Soc., 1952, lO94). However, fl-ionone is the most frequently used starting material since it already contains 13 of the 20 carbon atoms and is relatively easy to prepare from citral, which in turn can be either extracted from lemon-grass oil or synthesised from acetone (cf. Isler et al., 1961, loc. tit.). The extensive literature on vitamin A syntheses has been reviewed by Heilbron and Weedc n (Bull. Soc. chim. Fr., 1958, 83), Isler et al. (Vitamins and Hormones, 196o, i8, 295 ) and H. Pommer (Angew. Chem., I96O, 72, 811). (Concerning the introduction of x4C into various positions of the vitamin A skeleton, see Islet et al., loc. tit.). Vitamin A (A1) acid; retinoic acid. (I) Almost simultaneously with the publication of Isler's synthesis of vitamin A methyl ether (see p. 285), Van Dorp and Arens (Rec. Trav. chim., I946, 65, 338) announced the synthesis of vitamin A acid by a route contemporaneously explored by both Heilbron et al. (J. chem. Soc., 1946, 866) and Karrer et al. (Helv., 1946, 29, 704). The reaction sequence was as shown in Scheme 14 .

~

O

( i ) BPCH2 .CH'-"C H .CO 2M e/Zn (ii) - H 2 0

(ii) - H20

~

CO2H

Vitamin A acid ( ~ 1 1 )

Scheme t4. * Cf. p. 25o.

R

~

CO2Me

OHO LiMe

6

VITAMINS A

287

The sodium salt of the acid when buffered to pH IO was found to have activity comparable with that of vitamin A~ itself in the growth test; it is not reduced to vitamin A~ alcohol by the rat but is used as such (Arens and Van Dorp, Nature, 1946, I58, 622). Similarly, it is unable to alleviate eye disorders in A-deficient rats, for which the aldehyde (retinene) is required (J. E. Dowling and G. Wald, Vitamins and Hormones, 196o, x8, 515). Robeson et al. (loc. cir.) and Matsui et al., (J. Vitaminol., 1958, 4, 178) have since described syntheses of the all-trans, 9-cis, I3-cis, and 9,I3-dicis isomers of vitamin A acid (all of which were obtained crystalline); the vitamin A activity (growth test) of each has been measured (P. H. van Leeuwen et al., Nature, 1964, 2oz, 77)- The II,I3-dicis isomer (m.p. 128 ~ has also been synthesised (G. Pattenden et al., Chem. Comm., 1965, 347). (2) Vitamin A acid can also be obtained by oxidation (Tollen's reagent) of retinenel, which is itself readily obtainable from vitamin A 1 (see below; R. K. Barua and A. B. Barua, Biochem. J., 1964, 92, 2IC).

(b) Vitamin A 2: 3,4-dehydroretinol In 1937, Morton et al. and Heilbron et al. (Nature, 1937, x4o, 233, 234) reported that vitamin A concentrates obtained from certain fish liver oils gave an absorption band at 693 m# in addition to the normal (vitamin A1) band at 620 m# in the Cart-Price reaction (p. 282). The compound responsible for the 693 m/z band was named vitamin A 2 by Morton, the original vitamin A later being renamed vitamin A1. In general, liver oils from freshwater fish contain more vitamin A 2 than A x while those from marine fish contain more A 1 than A 2 (Heilbron, E. Lederer et al., Biochem. J., 1938, 32, 405). The difficulties associated with obtaining vitamin A 2 pure (in particular, free of vitamin A1) greatly hindered attempts to elucidate its structure; a pure sample (;tmax. 351 m/z) was eventually isolated from pike liver in 1948 by E. M. Shanlz (Science, 1948, io8,417). Meanwhile, during the period 1938-195o a variety of structures had been proposed for vitamin A~ (principally by Heilbron, Karrer, Morton and J. D. Cawley). Of these, the dehydrovitamin-A t structure (LXXXIII) seemed the most plausible to E. R. H. Jones et al. (J. chem. Soc., 1952, 2657) and this compound was, therefore, synthesised- vitamin A~ acid (synthetic LXXXII, cI. Van Dorp and Arens, loc. cit.) being used as starting material: LXXXII

_

(i) CH2N 2 (ii) NBS ;org. base ( i i i ) OH 0

~, / ~

A

t J.

_

(LXXXIV)

! [

.C02H

288

THE CAROTENOID

_CH2N2;LiAtH4

GROUP

~

7

.CH2OH

The product ( L X X X I I I ) had properties identical with those of Shantz's natural vitamin A 2 (so that L X X X I V is vitamin A2 acid).

Other methods of synthesis (I) From relatively accessible vitamin A] - by oxidation to vitamin A] aldehyde (LXXVI), bromination and then dehydrobromination (as above) to vitamin A 2 aldehyde, and finally reduction with lithium aluminium hydride (Jones et al., J. chem. Soc., 1955, 2765). (2) Isler et al. (Helv., 1962, 45, 517, 528) have reported total syntheses of all-tram vitamin A 2 (LXXXlII; obtained crystalline for the first time) and also of its I3-cis, 9-cis, 9,I3-dicis, II,I3-dicis, and II-Cis (LXXXV) stereoisomers, all of which except the last being obtained crystalline; the II,I3-dicis and II-Cis isomers contain hindered (cf. p. 250) double bonds. The methods used resemble those used in the technical synthesis of vitamin Aj (p. 285) except that here the starting material is the dehydro-fl-C]4aldehyde (LXXXVI) (idem, ibid., 1956, 39, 259), rather than the fl-C14aldehyde (XlX) 9

(LXXXVI)

( cf. LXXVII)

J AcCl, H| - H20), OHO, H2

(LXXvXV)"(11-cls )

I H2, AcCl, I2, H|

H20),OHO

(LXXXIlI) (alt-trans)

Vitamin A~, m.p. 63-650, (and also analogous compounds containing a cyclohexadiene ring) is very sensitive to atmospheric oxidation; it has only recently been obtained crystalline (see above). It shows 3o-4o% of the activity of vitamin A 1 (cf. Jones et al., loc. cit.).

(c) Retinene 1 and retinene 2 (retinal and 3,4-dehydroretinal) Retinene 1 and retinene2 were, respectively, the names given by Wald (J. gen. Physiol., 1938-1939, 22, 391) to carotenoid-like materials which he first detected in the retinas of frogs (ibzd., 1935-1936, 19, 351, 781) and certain freshwater fish (Wald, I938-I939,Ioc. cir.)" retinene x is also found else-

6

VITAMIN S A

289

where in nature, e.g. in leaves, rose hips, eggs (A. Winterstein and Hegediis, Z. physiol. Chem., 196o, 32x, 97; Plack, Brit. J. Nutrit., 1963, x7, 243). In 1948, Morton and his collaborators (Biochem. J., 1948, 42, 516) reported that vitamin A, could be oxidised by manganese dioxide in high yield to the corresponding aldehyde, and showed that this aldehyde had spectroscopic properties (ultraviolet spectrum; Carr-Price reaction) identical with those of Wald's retinene~ : ~

,

,

~

~

~

CH20H

Vitamin A I

CHO Retinene I

Later vitamin A z aldehyde was prepared similarly from vitamin A s ( L X X X I I I ) , and was found to resemble Wald's natural retinene 2 (Morton et al., Biochem. J., 1952, 52, 535; Jones et al., J. chem. Soc., 1952, 2657; 1957, 4909). Subsequently Robeson et al. (J. Amer. chem. Soc., 1955, 77, 412o) have shown that manganese dioxide will oxidise the synthetic (p. 286) 9-cis, I3-cis, and 9,I3-dicis isomers of vitamin A, to the corresponding cisretinenes 1 in high yield and without appreciable stereoisomerisation occurring, although a tendency for simultaneous oxygenation at C(4) has been observed (Jones et al., 1957, loc. cir.). W. Oroshnik (J. Amer. chem. Soc., 1956, 78, 2651) has similarly prepared (hindered) II-Cis-retinene, (see below). The cis-retinene% can be quantitatively reduced to the corresponding cis-vitamins A1 by t r e a t m e n t with potassium borohydride (P. K . Brown and Wald, J. biol. Chem., 1956, 222, 865): cis-vitamin A 1

MnOt

~ "

- cis-retinene x

KBH4

The Visual Process. The retina of the eye contains two types of light receptorrods (for vision in dim light) and cones (for vision in bright fight and for colour vision).In the dark a highly photosensitive carotenoid-protein complex(rhodopsin or "visual purple") accumulates in the rods, whilst on illumination of the retina the rhodopsin (purple) is bleached, being broken down into aU-trans-retinenex (yellow) and the protein ("opsin"). Attempts to reproduce the first of these processes in vitro using natural opsin and various synthetic stereoisomers of retinene t (cf. above) have shown that only those retinene t isomers with a certain (bent) molecular shape will couple with the protein, these being the 9-cis and the (hindered*) i I-cis forms; of these it is the latter which actually occurs in the eye (R. Hubbard and G. Wald, J. gen. Physiol., I952/53, 36, 269; Brown and Wald, 1956, loc. cir. ; Watd, Vitamins and Hormones, I96O, x8, 417). The bleaching process thus involves the isomerisation of the protein-bound I I-Cis-retinenex to the aU-trans form which, now being of the wrong shape to be bound to the * Cf. p. 250.

290

THE CAROTENOID GROUP

7

protein, dissociates from it. (Regarding the intermediates in this process, cf. T. Yoshizawa and Wald, Nature, 1964, 2o1, 34o). The mechanism by which the all-trans-retinenel is isomerised to the i i-cis form is less clear, but probably involves all-trans-and II-Cis-vitamin A 1 (cf. S. Futterman, J. biol. Chem., 1963, 238, 1145). Cone vision also depends on retinene I but a different protein is involved. The visual process in most animals apparently involves retinene 1 (and vitamin At) but in freshwater fish its place is taken by retinene~ (and vitamin As) (Wald, 196o, loc. cir.; cf. Pitt and Morton, Ann. Rev. Biochem., 1962, 3I, 492).

(d) Kitol Kitol, a vitamin A-inactive compound, frequently accompanies vitamin A 1 in fish liver oils. It is a dimer of vitamin A 1 and yields the vitamin on pyrolysis (N. D. Embree and Shantz, J. Amer. chem. Soc., 1943, 65, 91o); it was obtained crystalline by Robeson et al. (Science, 1947, xo5, 436), and subsequently by Garbers, Weedon and co-workers (Chem. Comm., 1965, 588); m.p. I38-i390, 2(EtOH) 295 mp. Kitol is formed on exposing vitamin A 1 to sunlight (R. Kaneko, Reports Govt. Chem. Ind. Res. Inst., Tokyo, 1962, 57, 194, 2o3; cf. also Y. Omote, C.A., 1964, 60, 58o3) and probably has structure L X X X V I I (Garbers, Weedon et al., loc. cit.)[by comparison of U.V. and P.M.R. spectra with those of model compounds, supported by the degradative work of Kaneko (loc. cir.) and by the mass spectral measurements of C. Giannotti, B. C. Das and E. Lederer (Chem. Comm., 1966, 28)] :

c~-~

~CH20H

"~

Vitamin A s apparently behaves similarly, giving "kitol," (cf. Kaneko and Motohashi, C.A., 1964, 61, 6859d).

(e) Properties of vitamins A 1 and A 2 and related compounds (i) Physical properties T h e m.p.'s a n d light absorption d a t a for t h e all-tram forms of v i t a m i n A x a n d A 2 a n d for five of their cis stereoisomers, a n d also for t h e corresponding aldehydes (retinenes) a n d acids, h a v e been collected and c o m p a r e d b y Isler

6

VITAMINS A

291

et al. (Helv., 1962, 45, 548); vitamin A activities are also given. In addition P.M.R. data are recorded for most of the alcohols, aldehydes and methyl esters of the acids and I.R. spectra are recorded for the A 2 alcohols; (for the Aj alcohols cf. Robeson et al., J. Amer. chem. Soc., 1955, 77, 4111) 9 (ii) Chemical properties* (I) Reaction with acid. As might be expected, vitamin A 1 (LXXIV), an allylic alcohol, is very sensitive to acid. Brief treatment with anhydrous ethanolic hydrochloric acid causes a characteristic shift in the absorption spectrum, the single peak of vitamin A1 (at 325 m/z) being replaced by a three-banded spectrum (392, 371, 351 m/z, in ethanol) (Heilbron et al., Biochem. J., 1932, 26,1164). In I 9 4 3 - I 9 4 4 S h a n t z el al. (J. Amer. chem. Soc., 1943, 65, 9Ol) and E. G. E. Hawkins and R. F. Hunter (Biochem. J., 1944, 38, 34) isolated the product, m.p. 77-78~ and showed that it was a hydrocarbon with 0"4% of the activity of vitamin A 1. The retro structure L X X X V l I I now generally accepted agrees with the spectral properties and the observed hydrogen uptake but has not been conclusively proved: it is obviously derivable from vitamin A 1.

Anhydrovitamin AI (LXXXVIII)

Vitamin Aj is converted into mlhydrovitamin A I also by antimony trichloride in chloroform (Shantz et al., 1943, loc. czt.), and so both compounds give the same Cars-Price reaction (Am~.. 620 ml~: cf. p. 282). Variants of the above method of preparation have been described (F. J. Paracek and Zechmeister, J. Amer. chem. Soc., 1956, 78, 3188; Shantz, J. biol. Chem., 195o,

x82, 5~5). Prolonged treatment of vitamin Aj (or anhydrovitamin A~) with ethanolic hydrochloric acid gives a compound with a pentaene chromophore which was named isoanhytlrovitamin A 1 (Shantz et al., 1943, loc. cir.) but which is now known to contain an ethoxyl group (Oroshnik, Science, 1954, I 19, 660). Shantz (195o, loc. cir.) showed that the weak vitamin A activity of anhydrovitamin A 1 (in the rat) was due to its being partly converted by the animal into a relatively active compound which was isolated from rat liver and named rehydrovitamin A1; this substance contains a pentaene chromophore and one non-allylic hydroxyl group. Brief treatment of vitamin A~. with ethanolic acid, as above, produces a 9 Additional to the dehydrogenation, oxidation-reduction, and dimerisation reactions m e n t i o n e d on pp. 2 8 7 - 2 9 o .

292

THE CAROTENOID

GROUP

7

compound, m.p. 89- 5 ~ originally formulated as a hydrocarbon and called anhydrovitamin A 2 (Shantz, Science, 1948, xoS, 417) but now known to be 3-ethoxyanhydrovitamin Aj (LXXXlX) (Jones et al., J. chem. Soc., 1955, 2763). This also shows weak vitamin A activity in the rat being converted therein into a more polar compound (rehydrovitamin A2) which lacks the ethoxyl group (M. S. Bamji et al., J. biol. Chem., 1962, 237, 2747)"

[A compound isolated from a fish liver-oil by R. K. Barua and P. G. Naya~, (Biochem. J., 1966, ioi, 302) and tentatively identified as the hydroxyanalogue of L X X X I X is known as "naturally-occurring anhydrovitamin A 2"]. (2) Diels-Alder reaction, with maleic anhydride. All-trans-Vitamin A 1acetate when left in ether with a large excess of maleic anhydride at 25 o yields (24 hr.) a mono-adduct (m.p. 96~ which, from its absorption spectrum (2max. 261, 238 m#), has structure XC: r162

~~VO

~_

CH2C)Ac O

(XC:) Other esters, (e.g., palmitate, p-phenylazobenzoate) of both all-trans- and 9-cis-vitamins A 1 react similarly but esters of the I3-cis isomer react only very slowly (Robeson et al., J. Amer. chem. Soc., 1955, 77, 4111; 1947, 69, 136). At higher temperatmes (8o~ all-trans-vitamin A1 acetate reacts with maleic anhydride to give a di-adduct. (W. J. Serfontein et al., J. S. African chem. Inst., 1963, z6, 22). (3) Formation of adduct with iron carbonyl. Retinene 1 reacts with dodecacarbonyl tri-iron, Fe3(CO)I~, in benzene to give a mono-adduct, m.p. I42~ which has the following structure (spectra; X-ray crystallography: A. J. Birch et al., Chem. Comm., z966, 613):

~

Fe(CO)3

CHO

7

CYCLIC EPOXIDES

293

7. Carotenoid compounds containing oxygen; "xanthophylls"* (a) Cyclic carotenoid epoxides : general properties Treatment of a carotenoid having alicyclic end-groups with monoperphthalic acid leads to preferential attack of the terminal (ring) double bond with the formation of a 5,6-epoxide, thus with a moderate excess of per-acid fl-carotene gives the diepoxide XCI and lutein (p. 295), as its diacetate, gives the monoepoxide XCII (P. Karrer and E. Jucker, Helv., 1945, 28, 300, 427; cf. M. S. Barber et al., J. chem. Soc., 196o, 2870):

(XCI)

(XCII)

The acyclic compound lycopene reacts slowly and gives only a trace of the 5,6-epoxide (W. V. Bush and L. Zechmeister, J. Amer. chem. Soc., 1958, 80, 2991 ) . The characteristic reaction (Karrer and Jucker, loc. cit.) of the carotenoid epoxides is their very rapid rearrangement to thefuranoid oxide form in the presence of a trace of acid (e.g. by brief treatment with a dilute solution of hydrogen chloride in chloroform); a small amount (~ 5%) of the hydrocarbon is formed simultaneously (cf. p. 303; also Karrer and Jucker, Helv., 1945, 28, 471):

Probably

via

~a,,,.

[A furanoid oxide so produced can sometimes be resolved into two epimers (about Cs) by chromatography]. This reaction is accompanied by a spectral shift of 20-25 m/z for each epoxide group rearranged, so providing a convenient test. Carotenoid epoxides are normally unaffected by bases, but they undergo gradual iearrangement to the corresponding furanoid form on active (even acid-free) surfaces (e.g., during prolonged chromatography on alumina). When a solution of an epoxide (or furanoid oxide) in ether is * Cf. p. 234.

THE CAROTENOID GROUP

294

7

shaken with concentrated hydrochloric acid, the acid layer turns blue, the depth of the colour and its stability depending on the oxide tested; some polyene aldehydes and polyhydroxy-carotenoids also give blue colours (cf. Karrer, Fortschr. Chem. org. Naturst., 1948, 5, I). Using the above methods, the epoxides and furanoid oxides of many carotenoids have been prepared and several of these have been identified with carotenoids present in nature (see p. 295 et seq.). Naturally-occurring furanoid oxides are probably in vivo rearrangement products of the epoxides, the rearrangement being caused by plant acids [1% citric acid causes the rapid rearrangement of zeaxanthin diepoxide (violaxanthin) ; and on storing orange juice the epoxides initially present disappear and are replaced by furanoid oxides: A. L. Curl, J. agric. Food Chem., 1954, 2, 685; 1956, 4, 159]. The variations which occur in the violaxanthin, antheraxanthin and zeaxanthin levels in isolated green leaves (a) when illuminated in an oxygenfree atmosphere and (b) during subsequent exposure to oxygen in the dark, indicate that the following process operates (H. Y. Yamamoto, T. O. M. Nakayama and C. O. Chichester, Arch. Biochem. Biophys., 1962, 97, 168; cf. also M. S. Bamji and N. I. Krinsky, Fed. Proc., 1964, 23, 430)"

NO

~

OH"FAnthe~axanthin

Violaxanthin ( a - under-

N2

Zeaxanthin

in tight ; b - u n d e r " 0 2 in the dark )

This facile reversible loss of the epoxide oxygen atom suggests that carotenoid epoxides may be involved in oxygen transport in biological systems (or act as an "oxygen reservoir"). The complex changes which occur in the carotenoid composition of the leaves and fruits of the red and yellow peppers during ripening can be accounted for by invoking three processes (fl- -+ ~-carotene; zeaxanthin -+ lutein; fl- -+ ~-kryptoxanthin) oi the type (L. Cholnoky et al., Acta Chim. Acad. Sci. Hung., 1958, 16, 227):

(O)

0

"a' ( - 0 )

H O ~ - - -

HO %

"b' ( Re~rrt. )

(In red peppers, pathway 'b' replaces 'a' at maturity; p. 345).

' ~ ' ~ - ID,. '

7

CYCLIC H Y D R O X Y C A R O T E N O I D S

295

The formation of its epoxide p r o b a b l y represents the first stage in the oxidative degradation of a carotenoid in dying leaves (T. W. Goodwin, Biochem. J., 1958, 68, 503).

Chemical deoxygenation of carotenoid epoxides can be effected with propylmagnesium bromide in the presence of ferric chloride: t-carotene diepoxide gives ( ~ 7o%) t-carotene, and violaxanthin gives (~3o%) zeaxanthin (L. Jaeger and P. Karrer, Helv., 1963, 46, 683; cf. Cholnoky et al., Chem. Comm., 1966, 4o4, for an alternative method): ....

,,per-acid

-PrMgBr/FeCI3

Regarding the biosynthesis of epoxides, cf. p. 344.

(b) Cyclic hydroxycarotenoids and their oxides, and the hydrocarbon oxides* Lutein (sometimes called "xantkophyll" (cf. p. 234) or "leaf xanthophyll"). R. Willstdtter and W. Mieg (Ann., 19o7, 355, I) first isolated lutein in a crystalline state and showed it to be C40H560=. S u b s e q u e n t l y it was shown to contain eleven double bonds (by h y d r o g e n a t i o n : L. Zechmeister and P. Tuzson, Ber., I928, 6z, 2oo3), to h a v e the same c h r o m o p h o r e as =-carotene, and to contain two h y d r o x y l groups (Zerewitinoff d e t e r m i n a t i o n ) ; t h a t these were secondary was shown b y the formation of a diketone on oxidising p e r h y d r o l u t e i n ; in addition, a series of diesters could be prepared (P. Karrer et al., Helv., I93O, z3, 87, 268, 7o9, lO99; 1933, x6, 977)- Oxidation with p e r m a n g a n a t e gave (from the rings) ==dimethylsuccinic and d i m e t h y l m a l o n i c acids b u t no ~x-dimethylglutaric acid showing (cf. p. 244) t h a t t h e o x y g e n functions are a t position 3 in b o t h rings. L u t e i n ' s optical activity, lack of v i t a m i n A activity, conversion into z e a x a n t h i n on heating with sodium ethoxide in benzene (p. 258 ), and t h e isolation of a compound, =-citraurin, shown to h a v e s t r u c t u r e XClII, on mild oxidation with p e r m a n g a n a t e (cf. p. 259 ) (R. K u h n et al., Z. physiol. Chem., 1931, z97, 141; Karrer et al., Helv., 1934, z7, 24; 1947, 3o, 266; 1938, 2x, 445) are all consistent with K a r r e r ' s f o r m u l a t i o n of lutein as a d i h y d r o x y derivative of ~-carotene (Karrer et al., ibid., 1933, x6, 977):

Q-Citraurin (XCIII)

* Regarding the trans -+ cis isomerisation of these carotenoids, see the footnote on p. 266. M.p.'s quoted were generally determined in evacuated capillaries. The characteristic properties of the epoxides are described on pp. 293 et seq.

296

THE CAROTENOID

GROUP

7 OH

H

Lutein is one of the most abundant xanthophylls, occurring in the green parts of all plants and also in egg yolk. Lutein dipalmitate, helenien, m.p. 92 ~ occurs in many red and yellow blossoms (Kuhn and A. Winterstein, Naturwiss., I93O, I8, 754; cf. also K u h n et al., Z. physiol. Chem., 1931, 197, 141. For its preparation from lutein and palmitoyl chloride, see Karrer and S. Ishikawa, Helv., I93O, 13, 7o9, lO99). Physical properties. Lutein crystallises from methanol in red prisms (with one mole of methanol of crystallisation), m.p. 1930 (corr.)" [ x ] ~ + 1450 (ethyl acetate), +16o ~ (chloroform); Xmax. 476, 446, 420 (petrol); 5o8, 475, 445 m# (carbon disulphide) (Kuhn et al., Z. physiol. Chem., 1931, 197, 141, 161; Ber., 1931, 64, 326); for the absorption curve in hexane, see Karrer and E. Wiirgler, Helv., 1943, 26, 117 and for P.M.R. data, B. C. L. Weedon et al., J. chem. Soc., 196o, 287o. For the preparation of lutein diacetale, m.p. 17o~ and other esters, cf. Karrer and Ishikawa, loc. cit. ; and for the preparation of the two (isomeric) monomethyl ethers and also the dimethyl ether see Jensen and S. Hertzberg, Acta Chem. Scand., 1966, 2o, 17o3; Mi~ller and Karrer, Helv., 1965, 48, 291. Lutein-5,6-epoxide ( = xanlhophyll epoxide: cf. p. 234). According to Karrer and his collaborators, lutein epoxide is a common constituent of green leaves (Helv., 1945, 28, 1526; 1948, 31, 113; cf. also Fortschr. Chem. org. Naturstoffe, 1948, 5, 15) and of flowers (Helv., 1945, 28, 1146; 1946, 29, 1539; 1947, 30, 537, 1158, 1774); for its preparation from lutein, see p. 293. Karrer and E. Jucker (ibid., 1945, 28, 3oo) found t h a t on treating lutein epoxide with a dilute solution of hydrogen chloride in chloroform, two isomeric [probably epimeric, about C(8): (XCIV) ] colouring matters were formed and showed t h a t they were identical with two carotenoids which had previously been isolated from natural sources, viz., flavoxanthin and chrysanthemaxanthin: OH

HO Ftavoxanthin, chrysanthemaxarthin (•

Flavoxanthin, first described by K u h n and H. Brockmann (Z. physiol. Chem., 1932, 213, 192), is most readily isolated from buttercup or dandelion flowers (idem, loc. cit.; Karrer and J. Rutschmann, Helv., 1942, 25, 1144); it crystaUises from methanol in lustrous yellow prisms, m.p. 184 o (corr.), I78~ (uncorr.); ~nax. 478, 448, 42o m# (carbon disulphide); [~]ca + 19~ (benzene); flavoxanthin diacetate has m.p. 157 o (uncorr.). Chrysanthernaxanthin was isolated from chrysan-

7

CYCLIC H Y D R O X Y C A R O T E N O I D S

297

t h e m u m and broom flowers, m.p. I 8 4 - I 8 5 ~ (uncorr.; from methanol); 2max. : as for flavoxanthin; [a]Hg, C-line ~ + 190 o (benzene) (Karrer et al., ibid., I943, 26, 626; 1944, 27, 1585; 1945, 28, 3oo, 1156). Zeaxanthin was first isolated from maize (Karrer, H. Salomon and H. Wehrli, ibid., 1929, 12, 79o). Its structure followed from investigations similar to t h o s e used for lutein (cf. Karrer etal., ibid., I93O, 13, lO84; 1932, 15, 49o; 1933, 16, 977). In addition, the conversion of r h o d o x a n t h i n into zeaxanthin (p. 315) helped t o confirm the structures of b o t h compounds; subsequently zeaxanthin was synthesised (see below). OH

HO

Zeaxanthin (XCV)

Z e a x a n t h i n occurs widely in plants b o t h in the free state and as its dipalmitate, physalien, m.p. 99 ~ [a]Hg, C-]ine (CHC13)--3 ~ (Kuhn et al., Bet., 1934, 67, 596; 193 o, 63, 1489); the l a t t e r was first isolated, and in high yield, from .Physalis alkekengi and P./ranchetti b y K u h n and W. Wiegand (Helv., 1929, 12, 499; see Winterstein and U. Ehrenberg, Z. physiol. Chem., 1932, 2o7, 25, for other sources), and later was synthesised from zeaxanthin and palmitoyl chloride b y Kuhn and C. Grundmann (Ber., 1934, 67, 596). Zeaxanthin is most conveniently isolated from maize or b y saponification of physalien (from Physalis spp. above); it also occurs, with lutein, in egg yolk (Kuhn, Winterstein and E. Lederer, Z. physiol. Chem., I93I , 197, I4I ) . Zeaxanthin shows no vitamin A a c t i v i t y (cf. H. yon Euler et at., Helv., 1934, 17, 24). Physical properties. Copper-red leaflets from m e t h y l e n e dichloride-methanol, m.p. 2o5-2o6 ~ (uncorr.); from benzene-methanol, m.p. 215 ~ (corr.); ~max. 48o, 452 (petrol) ; 517, 482 m/z (carbon disulphide) (0. Islet et al., ibid., 1956, 39, 2o41 ; Kuhn and Grundmann, loc. cir.; Zechmeister et al., Ber., 1939, 72, 1678); for t h e

3 Steps ~

3steps

X..o

I sophorone ~

Acetalisation [HC(OEt)3] ; LiAIH4 ;Ac20 ; Hr9 ~ v

~

0

CH2:CMe'OAc/H(~

AcO" v

AcO~ ~ ~ ~ O A c

Mild base

~

O

AcO" V (XCVl)

Scheme 15 .

298

THE

CAROTENOID

7

GROUP

absorption curve, and also I.R. spectrum, see Islet et al., loc. cir. ; for P.M.R. data, see Weedon et al., loc. cir.; [X]Hg, C-line - - 4 ~ (all-trans isomer, in chloroform; neo-A isomer, ~ + 12o o and neo-B, ~ o~ Zechmeister et al., loc. cir.). Synthesis. In 1956-1957, Islet and co-workers described several t o t a l syntheses of optically-inactive (probably racemic) zeaxanthin; oxygen was introduced into the carotenoid skeleton a t C(s) as above (Helv., 1956, 39, 2o41; 1957, 4o, 456) (Scheme I5). A Ca~ + C= + C19 --+ C40 scheme then gave zeaxanthin (Islet et al., 1957, loc. cir.) (Scheme 16; for the first step cf. p. 246 ). C 1-12= C H O E t ;MeCH ---CHOEt

(XCVl)

AcO

.~~Z) BrMgCmCMgBr (XCVlt)

OH

/OAc H~), OH@

Ar /OH Partial redn.; c l s - t r a n s isomerisn. =-

XCV

Scheme I6,

Other syntheses described b y Islet et al. include the (C144-C1, 4- C~4--*C4o) condensations shown in Scheme 17 (Ann., I957, 6o3, I29; C.A., I959, 53, I8982i, I8983f) 9 C~t (I) 2

+

ZnCIz/EtOAc; H 0 cf. p. 246

OEt (cf. XCVl )

(XCVIII)

0 i

~

I01

v

---

-

\v

\'f

I

~CLI

~OA/nndorf

~

A

cO

(xcIx)

POet3 / c~,H~ then OH el~ar~ial reduction~ e t c

Scheme I 7.

~- XCV

--

redn,;

then H| e ~

XCV

7

CYCLIC

299

HYDROXYCAROTENOIDS

The C1~ di-enol ether XCVIII was prepared from x-methylcrotonaldehyde and acetylenedimagnesium bromide; X C I X was prepared as follows:

CH2Br

PrNO2/OH O _ v

Wittig reaction with C

~ O

(c)

(cf. L X X V I I , p. 285). Zeaxanthin forms a diacetate, m.p. 154-155 ~ and a series of other di-esters (for the dipalmitate, see under "physalien" above) (Karrer et al., Helv., 1935, 18, 477; 1932, zS, 1195; 1934, 17, 55) and both a mono- and di-methyl ether (m.p.'s 153 o and 176~ (ibid., 1933, 16, 1163; H. Mi~ller and tfarrer, ibid., 1965, 48, 291). Perhydrozeaxanthin has [~]n (in CHC13) ~ J 3 o~ (Isler et al., ibid., 1956, 39, 2o41; t f u h n et al., Ber., I93O, 63, 1489). The 5,6-mono- and 5,6: 5',6'-di-epoxides of zeaxanthin were first prepared by Karrer and Jucker (Helv., 1945, 28, 3oo) by treating zeaxanthin diacetate with an ethereal solution of monoperphthalic acid, and they then realised that these two partially synthetic compounds were identical with two naturally-occurring carotenoids (antheraxanthin and violaxanthin) of, till then, unknown structure (see below). 4,4'-Dihydroxy-fl-carotene, commonly known as isozeaxanthin, has been synthesised (Islet et al., ibid., 1956, 39, 449; 1959, 42, 841; Entschel and Karrer, ibid., 1958, 41, 4o2; cf. p. 262) but has yet to be definitely identified in nature (cf. under isokryptoxanthin, p. 302). Antheraxanthin, m.p. 2o5 ~ ~aax. 5IO, 478 m/~ (carbon disulphide), was first isolated from the anthers of Lilium tigrinum and occurs as a cis form, m.p. I IO~ Amax. 506, 476 m/~ (CS~), in L. candidum (Karrer et al., ibid., 1935, z8, 13o3; 1949, 32, 5o; cf. also ibid., 1948, 31, 802). Its structure (Karrer and Jucker, 1945, loc. cir.) followed from its identification with "synthetic" zeaxanthin monoepoxide (cf. above): OH

HO

Anthera•

Antheraxanthin is probably the precursor of capsanthin (p. 323) in red peppers: p. 345. The furanoid oxide corresponding to antheraxanthin, mutatoxanthin probably occurs in orange juice, which is sufficiently acidic to rearrange epoxides (cf. P. 294) (A. L. Curl and G. F. Bailey, J. Agric. Food Chem., 1954, 2, 685). Mutatoxanthin, m.p. 177 ~ ~max. 456, 426 (petrol), 488, 459, 431 m~ (carbon disulphide), can be obtained in vitro by treating antheraxanthin or (mixed with auroxanthin:

300

THE CAROTENOID GROUP

7

cf. below) violaxanthin with hydrogen chloride in chloroform (Karrer et al., Helv., 1945, 28, 300; 1944, 27, 1684): OH

HO

MutatoxanthJn

Violaxanthin, first isolated from the petals of yellow pansies (Viola tricolor) by Ix'uhn and Winterstein (Ber., 1931, 64, 326), is now recognised to be one of t h e most a b u n d a n t carotenoids, being a c o m m o n constituent of green leaves (cf. p. 238 and also H. H. Strain, Arch. Biochem. Biophys., 1954, 48, 458) and flowers (e.g., marigold). I t was identified with " s y n t h e t i c " zeaxanthin diepoxide (cf. p. 299): ..OH

HO

9

9

Violaxanthin crystallises from m e t h a n o l - e t h e r in yellow prisms, m.p. 2000 (uncorr.), 2o7-2o8 ~ (corr.); Jtmax. 472, 442, 417 (petrol); 5o2, 469, 44 ~ m~u (carbon disulphide)" [ ~ ] ~ +350 (chloroform) (Karrer etal., Helv., 1945, 28, 3oo" I93I, x4, lO44; tfuhn and Winterstein, loc. cit.); regarding its biogenesis, cf. pp. 294, 344. W i t h dilute methanolic h y d r o g e n chloride, violaxanthin gives a m i x t u r e of a u r o x a n t h i n (see below), m u t a t o x a n t h i n and, a little, z e a x a n t h i n (XCV) (Karrer and Rutschmann, Helv., 1944, 27, 1684). W i t h alcoholic acetic or citric acids, deoxygenation to the l a t t e r two colouring m a t t e r s does not occur; instead two flavoxanthin-like colouring m a t t e r s (luteoxanthins a and b) (probably C(8) epimers of the intermediate epoxide-furanoid form of violaxanthin: cf. luteochrome p. 3o3) are formed (Curl and Bailey, loc. cir.; Strain, loc. cit.). Auroxanthin, m.p. 203 ~ /q-max. 428, 403, 382 m/z (ethanol), produced from violaxanthin as above, has the following s t r u c t u r e (Karrer and Jucker, Helv., 1945, 28, 3o0, 1156; and Karrer and Rutschmann, loc. cit.): oH

HO /

V

I\0

Auro~arlhin

Kryptoxanthin (fl-kryptoxanthin, cryptoxanthin) was first isolated in a pure state (from Physalis species) b y Kuhn and Grundmann (Ber., 1933, 66, 1746) who showed t h a t it differed from r-carotene only in the presence of a single O H group (active h y d r o g e n test; formation of a monoacetate) which t h e y placed at C(3 ) (by analogy with the position of oxygen functions in other carotenoids then known). This gave as the probable structure:

7

CYCLIC HYDROXYCAROTENOIDS

301

VO

and this has since been proved correct by total synthesis (see below). Kryptoxanthin has also been isolated from various fruits (including red peppers), maize, and egg yolk; papaya is a good source (C. Subbarayan and H. R. Cama, Indian J. Chem., 1964, 2, 451). It is a potent provitamin A having 60% of the activity of r-carotene itself (H. J. Deuel et al., Arch. Biochem., I950, 25, 6I).

Physical Properties. Lustrous

red leaflets from ether-methanol, m.p. I59 ~

(uncorr.); prisms from benzene-methanol, m.p. 1690 (corr.) (Islet et al., Helv., 1957, 40, 456; K u h n and Grundmann, loc. cit.); Amax. 519, 483, (452) m/~ (carbon disulphide); Islet et al., who also give the actual absorption curve, quote /~max. 480, 452 m/~ (petrol) and report on its optical activity and its I.R. absorption curve. Kryptoxanthin acetate can exist in two different forms, m.p.'s IiO ~ and 14o~ (L. Cholnoky and J. Szabolcs, Naturwiss., 1959, 46, 424); with per-acid the unsubstituted ring is attacked first (H. v. Euler et al., Helv., 1947, 3o, 1159). Synthesis. - By Islet et al. (ibid., 1957, 4o, 456) using a C19 + C,t --+ C40 route. The /~-C~,-aldehyde (XX; p. 246 ) was condensed with lithium acetylide to give the /~-C,t-carbinol (CI). This was then converted into kryptoxanthin in high overall yield using the acetoxy-/~-Cl,-aldehyde (XCVII) mentioned previously (p. 298) (Scheme 18). [3-Kryptoxanthin-5",6"-epoxide was (probably)detected by Cholnoky and his co-workers (Acta Chim. Acad. Sci. Hung., 1958, x6, 227) in unripe yellow peppers

ACO"

v

" (XCVII)

IEtMgBr OH

AcO

I H ; OHe

HO

~ Partiat redn.; isomerisation Kryp t oxanthin

Scheme 18.

(CI)

302

THE CAROTENOID GROUP

7

and by Curl and Bailey in oranges (J. Food Sci., 1961, 26, 442). The furanoid form of the 5,6-epoxide, k(c)ryptoflavin, has been detected ill papaya fruit (Subbarayan and Cama, 1964, loc. cit.), fl-Kryptoxanthin-5,6: 5",6"-diepoxide and the corresponding di-furanoid oxide, kryptochrome, can be prepared from flkryptoxanthin acetate using the methods outlined on p. 293 (Karrer and Jucker, Helv., 1946, 29, 229). Isokryptoxanthin. 4-Hydroxy-fl-carotene, synthesised by R. Entschel and Karrer from fl-carotene (ibid., 1958, 4 I, 983: cf. p. 263), is commonly known as isok(c)ryptoxanthin; it has as yet been only rarely (and tentatively) detected in nature (ci. e.g., W. L. Lee, Comp. Biochem. Physiol., I966, z9, I3). ~-Kryptoxanthin was the name given by Cholnoky et al. (Ann., 1958, 616, 207) to a colouring matter, m.p. 182 o (corr.), 2max. 478, 448, 42o (petrol), 509, 477, 446 mp (carbon disulphide), [X]Hg-C + 3600 (benzene), which they isolated from yellow peppers; it gives a monoacetate, which exists in two forms (cf. fl-kryptoxanthin acetate), m.p.'s 114-116 ~ and 148-15 ~ (Cholnoky and J. Szabolcs, Naturwiss., 1959, 46, 424). x-Kryptoxanthin's properties suggested t h a t it has the following structure:

HO

q - Kryptoxanthin

(?)

According to Cholnoky et al. (1958, loc. cir.) the biogenetic sequence fl-kryptoxanthin ---> fl-kryptoxanthin-5",6"-epoxide --+ x-kryptoxanthin, is one of three similar sequences which operates in ripening peppers (cf. p. 294). "Physoxanthin", m.p. 1530 (uncorr., from hot ethanol), [X]Hg-C + 290 o (benzene), a colouring m a t t e r isolated by C. Bodea and E. Nicoara (Ann., 1957, 6o9, 181 ; 196o, 635, 137) from the sepals of Physalis alkekengi, which at one time was also assigned the above structure by Bodea and Nicoara, has since been identified (Cholnoky et al., ibid., 1963, 663, 2o2) with a cis isomer of fl-kryptoxanthin (neofl-kryptoxanthin A), which has m.p. 76o (corr.), [~]Hg-C + 2 8 ~ (benzene), when crystallised from benzene-methanol in the cold but which gives a mixture of cis-trans isomers, m.p. i56~ when crystallised from hot aqueous ethanol. x-Carotene-5,6-epoxide, m.p.175 ~ ~max. 471, 442 m/~ (petrol), was prepared by Karrer and Jucker (Helv., 1945, 28, 471) from a-carotene (p. 271) by t r e a t m e n t with per-acid; it has been isolated from certain flowers either as such or as its furanoid derivative, flavochrome, m.p. 189 ~ ~max. 45 O, 422 m/~ (petrol) (Karrer et al., ibid., p. 1146). a-Carotene epoxide (but not flavochrome) has vitamin A activity (cf. p. 283):

Ftavochrome

7

CYCLIC HYDROXYCAROTENOIDS

303

fl-Carotene mono- and di-epoxides, and their /uranoid derivatives. Treatment of fl-carotene with 1.5 moles of perphthalic acid in ethereal solution gives a mixture (separable on calcium hydroxide) of fl-carotene-5,6-monoepoxide, m.p. 16o ~ uncorr., fl-earotene-5,6: 5',6'-diepoxide, m.p. I84 ~ uncorr., and the partial furanoid rearrangement product, luteochrome, m.p. 176~ uncorr., ~max. 511, 479; 5o2, 472; and 482, 45x m/~ (carbon disulphide), respectively (Karrer and Jucker, ibid., p. 427; cf. also K. Tsukida and Zechmeister, Arch. Biochem. Biophys., 1958, 74, 4o8) 9 On dissolution in dilute chloroformic hydrogen chloride solution, the monoepoxide yields its furanoid derivative, mutatochrome and a tittle ~-carotene (by de-oxygenation: cf. p. 293); the diepoxide yields aurochrome ( ~ 25~/o), mutatochrome ( ~ 25%: by partial deoxygenation), and ~-carotene ( ~ 5%) (Karrer and Jucker, loc. cit.; cf. Weedon et al., J. chem. Soc., 196o, 287o ). A urochrome crystallises from benzene-methanol in lustrous yellow leaflets, m.p. 185 o (uncorr.) and mutatochrome in orange leaflets, m.p. 163-164 ~ (uncorr.); AAmax. 457, 428 and 489, 459 (carbon disulphide); 426, 4ox, 38o and 453, 427, 404 m/~ (hexane), respectively (Karrer and Jucker, loc. cit.; Tsukida and Zechmeister, loc. cir.). ~-Carotene mono-epoxide probably occurs as such in yellow peppers (Cholnoky et al., Acta Chim. Acad. Sci. Hung., 1958, I6, 227). In addition in 1947, Karrer and .fucker (Helv., 1947, 3o, 536) identified a colouring matter, "citroxanthin", they had earlier isolated from orange peel (ibid., 1944, 27, 1695) with mutatochrome. J. Tischer's "flavacene" (Z. physiol. Chem., 1939, 260, 257) may also have been mutatochrome.

/3- Car'otene monoepoxide

Mutatochrome

Aurochrome

Rubixanthin, m.p. 16o 0 (red needles from benzene-methanol) and '~max. 494, 462, 432 (hexane), 533,494, 461 m/~ (carbon disulphide), was first isolated from rose hips (Kuhn and Grundmann, Bet., 1934, 67, 339, 1133); it is of limited occurrence being mainly found in various species of rose. It has the same

304

THE CAROTENOID GROUP

7

chromophore as y-carotene, absorbs I2 moles of hydrogen on hydrogenation, gives one mole of acetone on ozonolysis but shows no vitamin A activity, and contains one OH group (Zerewitinoff). Jensen et al. (Acta Chem. Scand., 1964, x8, 17o3) have shown t h a t the OH is not allylic to the chromophore (chloroformhydrogen chloride test: cf. p. 264) and, G~ the two remaining locations, C(s ) is by far the more likely (by analogy with other hydroxy-carotenoids) :

Rubixanthin (probably)

Rubixanthin-5,6-epoxide, m.p. (of monohydrate) 17o-17 I~ /~max. 526, 49x, 461 m/~ (carbon disulphide), and its furanoid derivative, rubichrome, m.p. I540, 2max. 5o6, 476 m/~ (carbon disulphide), have been isolated from the flowers of Tagetes patula by Karrer and co-workers (Helv., I947, 30, 531). Gazaniaxanthin, the major carotenoid of the flowers of Gazania rigens (K. SchOn, Biochem. J., I938, 32, I566; Zechmeister and Schroeder, J. Amer. chem. Soc., 1943, 65, I535), has m.p. I36-i370 and appears below rubixanthin on chromatography but closely resembles it in all other properties: gazaniaxanthin may be a cis isomer of rubixanthin. Saproxanthin, isolated from Saprospira grandis (an aquatic micro-organism) by A.J.Aasen and Jensen (Acta Chem. Scand., I966, 2o, 8ii), has m.p. I78-I79 ~ 2max. 500, 470, 445 m/~ (petrol), and probably has the following structure (spectra; partition behaviour; no allylic OH present; gives a monoacetate in the cold and this on treatment with phosphorus oxychloride/pyridine yields a product with an additional conjugated double bond and which is apparently identical with one of the products obtained on treating rubixanthin acetate with N-bromosuccinimide). OH H Sapr'oxanthin (probably) Celazantl~u, a hydroxylic carotenoid isolated by A. L. LeRosen and Zechmeister (Arch. Biochem., 1943, z, I 7) from the berries of Celastrus scandens, has m.p. 2o9-21o 0 (corr.), 2m~x. 562, 52z, 487 m/~ (carbon disulphide), and is possibly 3-hydroxytorulene (cf. Winterstein et al., Ber., 196o, 93, 2951) :

Ho

i'-ttydroxy-z',2"-dihydro-~-earotene, 2max. 488, 459, (438) m/~ in petrol, occurs along with three structurally related compounds in the bright red bacterium

7

CYCLIC HYDROXYCAROTENOIDS

305

Mycobacterium phlei strain Vera; for references to structure determination and total synthesis see p. 317.

1'- Hydroxy-l'

2 ' - d ih y d n o - - ~ - - c a c o t e r i e

OI-I-Chlorobactene, ~-max. 491, 46o, 435 m/~ (petrol), accompanies chlorobactene (p. 279) in three species of photosynthetic green bacteria investigated by Jensen, E. Hegge and L. M. Jackman (Acta Chem. Scand., 1964, z8, 17o3). The presence of a tertiary non-allylic hydroxyl group was inferred from polarity tests and its negative response to acetylation conditions and to chloroformhydrogen chloride. On t r e a t m e n t with phosphorus oxychloride in pyridine (5o~ 3o rain.), the colouring m a t t e r was converted in high yield into the known compound chlorobactene, so establishing its structure as:

OH - ch/or'obactene (C:U3

Confirmation of this was provided by a total synthesis (two-stage Wittig reaction) of compound CII by R. Bonnett, A. A. Spark and Weedon (ibid., 1964, z8, 1739) and comparison of natural and synthetic colouring matters:

~C |

HzPPh 3 Br |

1.120/H~

CHzPPh3

(i) BuLl (ii) ! moteXXIV-

(p. 248)

(Wittig reagent derived from XLV, p. 267)

H

O

-.

~

cTr

(cf. p. 278).

Eschscholtzxanthin, first isolated by Strain (J. biol. Chem., 1938, z23, 425), is the major colouring matter in the petals of the Californian poppy (Eschscholtzia cali/ornica). I t has m.p. 19o~ (uncorr.), Amax. 5o2, 472, 442 (petrol), 542, 5o7, 474 m # (carbon disulphide), [=]66~s + 2250 (chloroform), and is rather sensitive to aerial oxidation; a series of di-esters has been prepared (Strain, loc. cir.; Karrer and E. Leumann, Helv., 1951, 34, 445). Structural investigations by Strain were extended by Karrer and Leumann who showed t h a t eschscholtzxanthin (C40H540,) lost both oxygen atoms on t r e a t m e n t with dilute chloroformic hydrogen chloride to give a hydrocarbon ("anhydroeschscholtzxanthin",

306

7

THE CAROTENOID GROUP

m.p. 196 ~ uncorr.) which absorbed at ~ 3 ~ m/~ longer wavelength [~.max. 578, 539, 5o3 m/~ (carbon disulphide)]. This observation coupled with hydrogenation and spectral data (12 double bonds, all conjugated) led Karrer and Leumann (lot. cir.) to propose the following structure (ClII); this was subsequently confirmed by its partial synthesis from zeaxanthin dipalmitate (see p. 315): OH

H

Anhydroeschscholtzxanthin (XXXVll)

(c) A cyclic hydroxy-, methoxy- and methoxyketo-carotenoids Lycoxanthin, m.p. 1680 (corr., violet needles from carbon disulphide-ethanol), ;tmax. 5o3, 472, 443 (petrol), 547, 507, 473 m/~ (carbon disulphide), and lycophyll m.p. 179 o (corr., violet leaflets from benzene-methanol), Amax. 5o4, 473, 444 (petrol), 546, 506, 472 m/~ (carbon disulphide), were first isolated in 1936 from the ripe berries of Solanum dulcamara (woody nightshade), and were detected also in tomatoes (Zechmeister and Cholnoky, Ber., 1936, 69, 422). The colouring matters analysed for C40H560 and C40H560,, respectively, had the same absorption spectra as lycopene (p. 266), and gave, in the cold (indicating non-tertiary OH), a monoacetate, m.p. 135 o (corr.), and a dipalmitate, m.p. 1790 (corr.), respectively, and were assigned the following (probable) structures:

HO

Lycoxan

,n

(.)

oH

Both have only rarely been reported other than in the sources above (and of the few allusions to lycoxanthin, several probably refer to the rather similar compound rhodopi~: see below).

7

CYCLIC HYDROXYCAROTENOIDS

307

Carotenoids /rom purple bacteria. The following carotenoids have all been isolated from (or detected in) a few members of the classes of purple bacteria (Chromatium, Rhodospirillum and Rhodopseudomonas spp.) ; regarding their biogenesis, see pp. 336 et seq. Rhodopin was first isolated from mixed cultures of purple bacteria (probably Rhodopseudomonas spp.: S. L. Jensen, Acta Chem. Scand., 1959, 13, 842) by Karrer et al. (Helv., 1935, z8, 13o6), who showed (ibid., 1936, 19, IOI9; 1938, 2I, 454) that it contains twelve double bonds and one OH group which resists acetylation and, therefore, is probably tertiary. Goodwin et al. (Arch. Mikrobiol., 1956, 24, 305,313) later isolated the same compound from various other purple bacteria but concluded that it was identical with lycoxanthin (above) and reported it then and later (cf. Nature, 1958, I82, 477) as lycoxanthin. However, Jensen (loc. cir.; Acta Chem. Scand., I959, x3, 2142) later confirmed that whereas lycoxanthin is readily acetylated, rhodopin is not, and proposed for it the following structure. Proof has been provided by Weedon and co-workers (ibid., I964, I8, 1739 ) who synthesised the compound (using a route similar to that used for OHchlorobactene, p. 305) and carried out a direct comparison of the synthetic material with natural rhodopin. Thiospirillum ]enense is a good source of natural rhodopin (K. Schmidt, Arch. Mikrobiol., 1963, 46, 127):

Rhodopln

Rhodopin has m.p. 171~ (from carbon disulphide-petrol), ~max. 5 ol, 470, 44 ~ (petrol); 547, 5o8, 478 m/z (carbon disulphide) (Karrer et al., 1935, 1938, loc. cit.). The corresponding I,I'-dihydroxy compound, "OH-rhodopin", has been detected in Rhodomicrobium vannielii (L. Ryvarden and Jensen, Acta Chem. Scand., 1964, z8, 643 ). 3,4-Dehydrorhodopin, m.p. 186-19o ~ Rmax. 517, 483, 455 m/~ (petrol), isolated from a strain of Rhodopseudomonas palustris by L. M. Jackman and Jensen (ibid., 1961, x5, 2o58), was shown to have the following structure from a consideration of its P.M.R. and visible absorption spectra: H

3,4- Dehydr'orhodopin

Pigments "P48z" and "OH-P481". In I956, Goodwin and D. G. Land (Arch. Mikrobiol., 1956, 24, 3o5) detected a colouring matter in a Chromatium sp. which had maxima in petrol at 514, 48x, 454 m/~ and designated it P48I ; it was (probably) also detected in other purple bacteria (Goodwin and Land, loc. cir.). The Chromatium also contained a second colouring matter which was rather more polar than P48I but had an identical absorption spectrum; it was designated

308

THE CAROTENOID GROUP

7

OH-P48I. The marked fine structure of their absorption curves and the position of the maxima indicated t h a t both P48I and OH-P48I have an acyclic dodecaene chromophore. ]ensen (Acta Chem. Scand., I958, zz, I698; I959, z3, 2143) found t h a t both substances contain one methoxyl group (modified Zeisel determination), which (according to their I.R. spectra: cf. p. 256) is tertiary and confirmed that OH-P48I, m.p. I9o.5 ~ ;tmax. (pure solid) 516, 483, 455 m/~ (in petrol), contains a tertiary hydroxyl group (by I.R. and its resistance to acetylation), and also t h a t neither the OH nor the OMe group is allylic (no reaction with hydrogen chloride in chloroform). Ozonolysis gave o . 6 mole of acetone, as expected (P. 244) for a molecule containing t w o - - C M e ~ O R groups. Of the two structures consistent with the above, the one (CIV) shown below and the isomer with the chromophore placed symmetrically, the former was favoured by Jackman and ]ensen (ibid., I96I, x5, 2058) on biochemical grounds. This conclusion has been confirmed by ]. D. Surmatis et al. (J. org. Chem., I966, 3x, I86) who obtained compound CIV by total synthesis and showed t h a t it is identical with OH-P48I isolated from purple bacteria; the synthesis involved a two-stage Wittig reaction to attach first one end-group then the other to the aldehyde groups of crocetindial (p. 248 ) (see the reaction schemes on pp. 309 and 305 for the methods used to prepare the end-group Wittig salts). OH-P48I is probably identical (cf. ]ensen, Acta Chem. Scand., I959, x3, 2143) with the pigment "rhodovibrin" described, b u t not obtained pure, by Karrer and U. Sotmssen in I936 (Helv., I936, x9, 3, IOI9): Me

H (CIV), Rhodovibrin ( --"OH-P481" )

P48I was tentatively assigned the following structure by Jensen (Acta Chem. Scand., I96I, z5, ii82, 2o58) ; this was confirmed by total synthesis by Surmatis et at. (1966, loc. cir.) using a route similar to t h a t used for OH-P48I above: Meo

"P 481" (=Anhydrorhodovibrin)

Spirilloxanthin (rhodoviolascin). Rhodoviolascin, isolated from'rhodovibriobacteria by Karrer and Solmssen (Helv., I935, z8, I3O6; I936, z9, IoIg), is identical (C. B. van Niel et al., Arch. Biochem., I944, 5, 243) with spirflloxanthin, the main carotenoid present in mature cultures of Rhodospirillum rubrum and also found in other purple bacteria (van Niel and J. H. C. Smith, Arch. Mikrobiol., z935, 6, 219; cf. ]ensen et al., Biochim. Biophys. Acta, I958, z9, 477; Phytochem., I965, 4, 925). Karrer et al. (Helv., I936, I9, 3; I94 o, 23, 46o) showed t h a t "rhodoviolascin" contains two methoxyl groups and I3 conjugated (acyclic) double bonds (hydrogenation; absorption spectrum) and isolated

7

ACYCLIC HYDROXY

309

ETC. C A R O T E N O I D S

bixindial (p. 327) and a higher (probably Cs0) dialdehyde on oxidation with permanganate. Its precise structure CV followed from its P.M.R. spectrum, which included signals due to six "in-chain" (z 8.o2), but no "end-of-chain", methyls, four methyls attached to carbon carrying oxygen (but not hydrogen) (3 8.83), two methyls attached to oxygen (T 6-78), and two --CH--CH~--- units (M. S. Barber, Jackman and Weedon, Proc. chem. Soc., 1959, 96): the same structure was inferred from chemical evidence by Jensen (Acta Chem. Scand., 1959, z3, 381): MeO~

Me Spiri[[oxanthin

(rhodovio[ascin)

(CV)

Spirilloxanthin has m.p. 2180 (uncorr., from benzene), ~max. 548, 511, 482 (benzene); 573, 534, 496 m # (carbon disulphide). Synthesis. Surmatis and A. O/her (J. org. Chem., 1963, 28, 2735) have described two routes both starting from methylheptenone (CVI) (used in vitamin A synthesis) and giving ~ lO% overall yield (Scheme 19).

(i)

BrCH2.CO2Et/Zn__-

Me "

CC~Et

NBS/CC[4 ~

(CVl)

Me<)

C02Et

LiAIH4; , Ph3P'HBr ~,, ~

MeO#

/CH2PPh 3 BPIS)

NaOMe/C2o-diat (~E[~r) , - ------ CV

(cvla) ~aOM~/XXW ( ii )

NBS/CCL 4

CVI

NBS = N-bromosuccinimide

----- CV

Scheme 19.

"OH-spirilloxanthln", a coloured substance detected in small amounts in Rhodospirillum rubrum (cf. Jensen et al., Biochim. Biophys. Acta, 1958, 29, 477) and since isolated crystalline (m.p. 209 ~ from Rhodopseudomonas gelatinosa (idem, Acta Chem. Scand., 1963, z7, 5oo), has spectral (~max. 528, 493, 461 m#, in petrol) and chromatographic properties expected of monodemethylated spirilloxanthin, a formulation supported (idem, 1963, loc. cir.) by its I.R. spectrum (bands assignable to tertiary OH and OMe} and its resistance to acetylation (see also below):

310

THE CAROTENOID GROUP

Me

7

H

(CVII}, Monodemethytated spirittoxanthin (= "OH- spiritto• ?)

Bacterioruberin at is, probably, the corresponding dihydroxy-compound (cf. Jensen, ibid., 196o, x4, 95 o, 953, who claims to have effected the conversion: bacterioruberin ~ --+ CV + CVlI by using methyl iodide-silver oxide in dimethylformamide). Chloroxanthin, isolated in 1958 from a green m u t a n t of Rhodopseudomonas spheroides (T. O. M. Nakayama, Arch. Biochem. Biophys., 1958, 75, 352) and later found in R. gelatinosa (Jensen, Acta Chem. Scand., 1963, 17, 5oo), has m.p. 139 o (corr., from petrol) and Amax. 468,438, 413 m/~ (petrol) (as for neurosporene, p. 276 ). Preliminary structural work was carried out by Nakayama (loc. cir.). Later, Weedon and his co-workers examined the P.M.R. spectrum of the compound, proposed the following structure, and subsequently confirmed this by total synthesis (Proc. chem. Soc., 1961, 261; Tetrahedron Letters, 1964, 2603):

H

Chtoroxanthin

Spheroidene ("pigment Y") and spheroidenone ("pigment R"), isolated from Rhodopseudomonas spheroides by van Niel (Antonie v. Leeuwenhoek [Amsterdam], 1947, 12, 156; cf. Goodwin, Arch. Mikrobiol., 1956, 24, 313 for further sourcesl, have m.p.'s of 1450 and I63 ~ respectively (corr., from petrol) and 2;-max. 486, 455, 429 and 512, 481, 459 m/~, respectively, in petrol (Nakayama, Arch. Biochem. Biophys., I958, 75, 356; Jensen, Acta Chem. Scand., 1963, 17, 5oo/. Goodwin, Land and M. E. Sissins (Biochem. J., 1956, 64, 486) established the nature of their chromophores and found t h a t each compound contained one methoxyl group; their full structures followed from the determination of their P.M.R. spectra (cf. Weedon et al., I961, loc. tit.), and were confirmed subsequently by total synthesis:

Me Spheroidene (' pigment Y ' )

Spheroidenone ('pigment R" )

7

ACYCLIC

HYDROXY

311

ETC. CAROTENOIDS

Synthesis (M. S. Barber et al., J. chem. Soc. [C], 1966, 2166): " Y " - by a Wittig reaction between CVla (p. 309) and the necessary Cs0-aldehyde , itself synthesised (4 steps) from the "iarnesyl Wittig reagent" (LXlIIa, p. 275); "IR" - Me=C(OMe).COC1 + C H = = P P h s --+ Me,C(OMe).CO.CH--PPh s (3steps) Me=C(OMe) -CO .CH = CH-CMe = CH. C H - - C H . CMe= CH. CH = P P h s (used also on p. 312); a Wittig reaction with the necessary C=6-aldehyde (itself synthesised by a I :I Wittig condensation of L X l I I a , p. 275, and the C10-trienedial derived from XXVI, p. 248 ) gave "'R". (Regarding the in vivo transformation of pigment Y into pigment R, cf. p. 344). OH-spheroidene ("0H-Y"). In 1963, Jensen (Acta Chem. Scand., 1963, 17, 500) isolated from Rhodopseudomonas gelatinosa, a coloured crystalline compound ("OH-Y"/ (m.p. I57 ~ from acetone-petrol), earlier detected therein by Goodwin (1956, loc. cir.), which had the same chromophore (Amax. 486, 454, 429 m#, in petrol) as, but was more polar than, spheroidene ("Y", above). Jensen confirmed that the compound contains a tertiary hydroxyl group, and (by I.R. spectrometry: p. 256) also a tertiary methoxyl group, and tentatively assigned it the following structure, which was later confirmed by an examination of the P.M.R. spectrum and by showing t h a t treatment with phosphorus oxychloride/pyridine yields spheroidene (Jackman and Jensen, Acta Chem. Scand., 1964, 18, 14o3):

MeO

OH

"OH- spheroidene"

OH-spheroidenone ("OH-R"). Goodwin (1956, loc. cir.) discovered that the pigments " Y " and " R " in certain Rhodopseudomonas spp. are accompanied by a substance spectrally similar to, but more polar than, " R " which was, therefore, designated " O H - R " . Jensen (Acta Chem. Scand., 1963, 17, 489) isolated " O H - R " crystalline from R. gelatinosa, m.p. 158-1590 (uncorr., from acetone-petrol), ~max. 516, 483, 46o m# (petrol: no fine structure in ethanol). The substance's visible and I.R. spectra, its negative response to acid, and its response to treatment with lithium aluminium hydride and then acid (cf. pp. 245, 256/, were all consistent with the structure shown, which was later proved to be correct by examining the P.M.R. spectrum and by showing t h a t treatment with phosphorus oxychloride/pyridine produces spheroidenone (Jackman and Jensen, 1964, loc.

cir.):

"OH

- spheroidenone"

Pigment P518, isolated from both Rhodopseudomonas spheroides and R. gelatinosa by Jensen (ibid., 1963, 17, 303, 489), has m.p. 222 ~ (dark violet needles,

312

THE CAROTENOID GROUP

7

from acetone-petrol), Rmax. 555, 518, 487 ( p e t r o l - no fine s t r u c t u r e in ethanol), 6Ol, 562, 528 m/z (carbon disulphide). Visible and I.R. spectra showed t h a t this substance was also a polyene ketone and t h a t it contained methoxyl, b u t not hydroxyl, groups. Small-scale chemical tests (as for OH-spheroidenone, above) and an e x a m i n a t i o n of its P.M.R. s p e c t r u m led to the structure shown below (Jackman and Jensen, 1964, loc. cir.), since confirmed b y direct comparison with synthetic 2,2'-dioxospirilloxanthin. Regarding its formation in vivo b y the oxidation of spirilloxanthin (CV), cf. p. 345 and Jackman and Jensen, loc. cit.; Goodwin's "P512" (1956, loc. cit.) was p r o b a b l y impure P518 (K. E. Eimhjellen and Jensen, Biochim. Biophys. Acta, 1964, 82, 21):

"P 518" (: 2,2"-dioxospirittoxanthin )

Syntheses: (i) B y an aldol condensation between the 15,I 5'-dehydro-derivative of the symmetrical C30-dialdehyde mentioned in the footnote on p. 248 and excess Me,C(OMe) .CO.Me, followed b y partial reduction etc. (cf. p. 247 ) (U. Schwieter et al., Helv., 1966, 49, 992); (ii) B y a W i t t i g reaction between two moles of the phosphorane Me,C(OMe) 9CO. CH = CH. CMe = CH. CH = CH. CMe = CH. CH = P P h 3 (cf. under spheroidenone, above) and the necessary symmetrical C10-trienedialdehyde (from X X V I , p. 248) (P. S. Manchand and Weedon, T e t r a h e d r o n Letters, 1966, 989). A substance, ~max. 544, 5Ix, 485 m/~ (petrol), detected in minute a m o u n t in R. gelatinosa b y Jensen (Acta Chem. Scand., 1963, 17, 555), had spectral and chromatographic properties corresponding to those expected for 2-oxorhodovibrin (cf. CIV), a proposed i n t e r m e d i a t e in the biogenetic p a t h w a y outlined on P. 337(d) A ldehydo-, keto-and hydroxyketo-carotenoids (i) Aldehydes and hydroxyaldehydes The following are in addition to the retinals discussed in connection with v i t a m i n A and its related compounds (pp. 288 et seq.). fl-Citraurin (citraurin) (C30), m.p. I47 ~ occurs in orange peel (L. Zechmeister and P. Tuzson, Bet., 1937, 7 o, 1966). Its s t r u c t u r e was established b y Karrer et al. (Helv., 1937, 2o, 682; 1938, 21, 4481 who noted its resemblance to fl-apo8'-carotenal (a compound which was already well known as a degradation p r o d u c t of r-carotene, p. 259) and later isolated it from the m i x t u r e obtained on mild oxidation of z e a x a n t h i n with p e r m a n g a n a t e (p. 259):

HO -

Cltraurin

7

ALDEHYDO-

AND HYDROXYALDEHYDO-CAROTENOIDS

313

/~-Apo-zo'-carotenal (C27) and /~-apo-8'-carotenal (C30) have been detected, by comparison with the synthetic compounds (p. 247), in citrus fruits, rose w

0

/3-Apo-8'- carotena[

hips, and grass; the corresponding C37- and C4o-aldehydes have similarly been detected in oranges and micro-organisms, respectively (A. Winterstein et al., Ber., 196o, 93, 2951) :

I

o

C,,-aldehyde (16'-oxotoru }ene)

Apo-6'-lycopenal (C32) and apo-8'-lycopenal (C30) occur in tomatoes (Winterstein

et al., loc. cir.). . . . . . . . . . .

"T

""

"T

"-

"~

A p o - 6 ' - [ycopenal

(ii) Keto- and hydroxyketo-carotenoids Rhodoxanthin was first detected in the leaves of Potamogeton natans by N. A. Monteverde and subsequently obtained crystalline by Monteverde and V. N. Lyubimenko (cf. C.A., 1914, 8, 913). I t also occurs in the red winter needles of various conifers (the " t h u j a r h o d i n " of M. Tswett, Compt. rend., 1911, 152, 788; T. Lippmaa, Ber. deut. bot. Ges., 1926, 44, 643) and in bird feathers (0. V61ker, Fortschr. Chem. org. Naturstoffe, I96O, 18, 183); yew (Taxus baccata) berries are a convenient source (Kuhn and Brockmann, Bet., 1933, 66, 828). Its structure C V l I I followed from its behaviour on hydrogenation (rapid u p t a k e of 12 moles of hydrogen, followed by slower uptake of a further 2 moles), the formation of a dioxime, and its absorption spectrum; confirmation was afforded by the observat-ion t h a t its dihydro-derivative (CIX: cf. p. 315) had the same absorption spectrum as fl-carotene (Kuhn and Brockmann) and could be converted (Karrer and U. Solmssen, Helv., 1935, zS, 477) into zeaxanthin. O .#

Rhodoxanthin (CVlli)

314

THE CAROTENOID

GROUP

7

Rhodoxanthin has been synthesised from zeaxanthin (p. 315) 9 in addition, a total synthesis has been outlined (R. Riiegg and G. Saucy, U.S.P., 2,983,752/1961" C.A., 1961, 55, 22369e)"

AcOH

AcO

( i ) KOH (ii) Oppenauer oxidn. (iii) Partial. redn., etc.

l

CVlll

Rhodoxanthin crystallises from benzene-methanol in violet needles, m.p. 2190 (corr.), Amax. 524, 489, 458 (hexane)" 564, 525, 491 m# (carbon disulphide)" its dioxime has m.p. 227-228 ~ (corr.), ~max. 513, 479, 451 m# (hexane). Crystalline rhodoxanthin is unusually resistant to atmospheric oxidation (Kuhn and Brochmann, loc. cit.). Eschscholtzxanthin (ClII, p. 3o6), rhodoxanthin (CVlII), and zeaxanthin (XCV, p. 297) have been inter-related as shown in Scheme 20. Eschscholtzxanthone, ~max. 479 m# (hexane), recently discovered (in small amount) in T. baccata (cf. rhodoxanthin, above), has been assigned the ketoalcohol structure intermediate between t h a t of eschscholtzxanthin and rhodoxanthin on the basis of its absorption, chromatographic, and partition properties (all intermediate between those of eschscholtzxanthin and rhodoxanthin), its I.R. spectrum, and the finding t h a t treatment with silver oxide yields rhodoxanthin (Bodea, E. Nicoara and T. Salontai, Rev. Roumaine Chim., 1964, 9, 517). In addition, it was shown t h a t the sequence rhodoxanthin --+ eschscholtzxanthone --+ eschscholtzxanthin could be carried out using borohydride (this also provides a partial synthesis of the compound from relatively accessible rhodoxanthin). Echinenone (myxoxanthin), first isolated by Lederer (Compt. rend., 1935, 2oz, 30o" cf. Bull. soc. chim. Biol., 1938, 20, 587) from Paracentrotus lividus (a sea urchin), is widely distributed in marine invertebrates (Goodwin et al., Biochem. J., I95 ~ 47,244, 249" Nature, 1951, 167, 358. J. Ganguly, N. I. Krinsky and J. H. Pinchard, Arch. Biochem. Biophys., 1956, 6o, 345). In addition, the colouring m a t t e r rnyxoxanthin, which has been isolated from many of the blue-green algae (Heilbron et al., J. chem. Soc., 1936, 1376" Goodwin, J. gen. Microbiol., 1957, z7, 467" Tischer, Z. physiol. Chem., 1958, 311, I4O), is identical with echinenone (a direct comparison of the colouring matters and their oximes was carried out

7

KETO-

AND HYDROXYKETO-CAROTENOIDS

315 9c o . c ~ m M m

C15H31.CO.

Zeaxanthin dipalmitate (i mild base (ii)) NBS;OH 0

,,~

(cIII) Eschscho tzxanthm

I ,,,,, 0

(iv) Zn/AcOH - pyr'id ine

0

0

OH

H9

(•

7ea•

Scheme 20.

Re/s. - K a r r e r et al., Helv., I958, 41, 402 [(i), (ii)]; I963, 46, 687 [(iii)]; K u h n and B r o c k m a n n , B e r . , I933, 66, 828 [(iv)] ; K a r r e r et al., Helv., I935, 18, 477 [(v)] ; I959, 42,

466 [(vi)]. * Ag20/CC14 is also effective: B o d e a et al., Rev. R o u m a i n e Chim., 1964, 9, 517, 839.

316

THE CAROTENOID GROUP

7

by Goodwin and M . M . Taha, Biochem. J., 1951, 48, 513). The structure of echinenone (CX), first inferred by Goodwin et al. (195o, loc. cit.) mainly from spectroscopic data, was defined chemically by Ganguly et al. (loc. cit.) who showed t h a t (a) reduction of echinenone with lithium aluminium hydride gave a compound identified with 4-hydroxy-fl-carotene and (b) Oppenauer oxidation of synthetic 4-hydroxy-fl-carotene (p. 264) gave (50%) a compound whose spectral and chromatographic properties were as for natural echinenone:

0

Echinenone (= myxoxanthin ) (CX)

In accordance with this formulation, echinenone shows ~ 5o% of the vitamin A activity of fl-carotene (Ganguly et al., loc. cit.). Echinenone crystallises from benzene-methanol in violet plates, m.p. 186-187 o (uncorr.); ~max. 456 (petrol); 472 m/~ (benzene) (one peak only: cf. p. 253 ) (Weedon et al., J. chem. Soc., 1958, 3986; 1959, 4o58; see also for I.R. data). Syntheses. (I) Ganguly et al. probably obtained echinenone from 4-hydroxyfl-carotene (above); (2) Petracek and Zechmeister (1956: see p. 261) isolated 4-oxo-fl-carotene in 5% yield from the mixture obtained on treating fl-carotene with N-bromosuccinimide in chloroform containing i % ethanol and identified it with natural echinenone; (3) The first rational synthesis was described by Warren and Weedon (ibid., 1958 , 3986). ~ - D i m e t h y l g l u t a r i c acid was converted (7 steps) into a methyl ketone CXI which on base-catalysed condensation with fl-apo-8'-carotenal (CXlI) (c~0, synthetic, cf. p. 247 ) gave (32 %) the decaenone (CXlII); two more steps furnished (4o%) echinenone (CX):

(CXII) KOH in EtOH I_ I (CXl)

'~ ?~]0

I P-M~'C6H4" S%H/aCet~ ~KOH in MeOH (cx)

7

KETO- AND HYDROXYKETO-CAROTENOIDS

317

(4) A more convenient synthesis, which utilises Robinson's Mannich-base reaction, is as follows (M. Akhtar and Weedon, ibid., I959, 4o58) :

0

l

~ KOH/EtOH

! (CXtl) I (Yield 8 0 % )

/\

o (t) I Et 3 N., + (CXlV) 0 (CX}

Yields were markedly improved (to 90% and 6O~/o, respectively) when the polyene aldehyde CXII was replaced by its I5,I5'-acetylenic analogue; the latter step proceeded in 45~o yield if the salt CXIV was replaced by ethyl vinyl ketone (CH2--CH. CO. Et) itself. The 15,I 5'-dehydroechinenone so produced was converted into all-trans-echinenone (CX) in the usual way (cf. p. 247). Aphanin. In 1938, Tischer (Z. physiol. Chem., 1938, 25z, lO9; 1939, 260, 257; cf. also ibid., 1944, 281, 143 ) isolated a keto-carotenoid (C40H540) from the alga Aphanizomenon flos-aquae and named it aphanin [m.p. 18o 0 (corr.), 2max. 505, 472 (benzene); 534, 494 m/~ (carbon disulphide)]. I t obviously resembled echinenone in m.p. and molecular formula, and Goodwin et al. advanced chemical (Biochem. J., 195 o, 47, 244) and biochemical (J. gen. Microbiol., 1957, z7, 467) arguments in favour of their i d e n t i t y , - an opinion subsequently confirmed by Hertzberg and S. L. Jensen (Phytochem., 1966, 5, 565) who carried out a direct comparison of aphanin freshly isolated from A. flos-aquae with synthetic echinenone [comparison of visible and I.R. spectra and m.p.'s; mixed chromatograms; comparison of the hydride reduction product of natural aphanin with synthetic (p. 302) 4-hydroxy-~-carotene]. "Aphanicin", also isolated by Tischer (1939, 1944, loc. cit.) from A. flos-aquae and reported to be a keto-carotenoid with m.p. I95 ~ 2max. 533, 494 m/~ (CS2), and alleged to be a provitamin A (Scheunert and Wagner, Z. physiol. Chem., 1939, 260, 272), has since been shown to be identical with canthaxanthin (vide in/ra; by comparison of spectra etc. as for aphanin, above: Hertzberg and Jensen, 1966, loc. cir.). 4-Oxo-7-carotene and I'-hydroxy-4-oxo-I',2'-dihydro-F-carotene, both with 2max. (49o), 465 m y (petrol), occur (along with 7-carotene and its I'-hydroxyI',2'-dihydro- derivative: p. 3o4) in Mycobacterium phlei Vera (idem, Acta Chem. Scand., 1966, 2o, 1187). Their structures were established by chemical interconversions and comparison (spectra; chromatographic comparisons of cis-trans mixtures obtained on iodine isomerisation) of the natural carotenoids, and of

318

7

THE CAROTENOID GROUP

selected derivatives, with the totally synthetic compounds (cf. A. P. Leflwick and Weedon, ibid., 1966, 2o, 1195). 2'

o 4-ox o- 2, - carotene Deoxy-flexixanthin, closely related structurally to the above compounds, with ~max. 503, 476 m/~ in petrol, has been isolated (along with its 3-hydroxy derivative, p. 322) from a Flexibacter species; its structure was deduced from spectral evidence and by the carrying out of a series of chemical interconversions (,4. J . Aasen and Jensen, ibid., 1966, 2o, 197o):

3

0

Deoxyf texixanthin

Canthaxanthin was isolated by F. Haxo (Bot. Gaz., 195o , IX2, 228) from the edible mushroom Cantharellus cinnabarinus and has since been found widely in bird feathers (0. V~lker, Naturwiss., 1961, 48, 581 ; D. L. Fox, Comp. Biochem. Physiol., 1962, 5, 31; 1962, 5, I) and elsewhere (S. Saperstein and M. P. Starr, Biochem. J., 1954, 57, 273; see also "aphanicin", above). In 1956, Petracek and Zechmeister (cf. p. 261) isolated (1%) a compound identified as 4,4'-di-oxo-~carotene from the mixture obtained on treating ~-carotene with N-bromosuccinimide in chloroform-ethanol and showed (Arch. Biochem. Biophys., 1956, fix, 137) t h a t it was identical with natural c a n t h a x a n t h i n (CXV) : o

Canthaxanthin has m.p. 213 o (corr., from benzene-methanol or chloroformethanol), 216-2170 (uncorr.) ; ;tmax. 466 (hexane or petrol), 478 (ethanol), 48o m/~ (benzene) (one peak only: cf. p. 253 ) (Petracek and Zechmeister, loc. cit.; Islet et al., Helv., 1959, 42, 841); for I.R. and P.M.R. data, see Weedon et al., J. chem. Soc., 1958, 3986; I96O, 287o. Syntheses. (I) By Petracek and Zechmeister, from ~-carotene in low yield: see above" (2) By Warren and Weedon, loc. tit., the first rational synthesis, using the m e t h y l ketone CXI used in their echinenone synthesis (p. 316) and crocetindial ( X X l V : cf. p. 248):

7

KETO-

AND

319

HYDROXYKETO-CAROTENOIDS O H @. H 9 O H @ (as on p. 316)

2 CXI + XXlV

+ CXV (15% overall)

(3) B y Akhtar and Weedon (loc. cir.) b y condensing 2 moles of isopropyl m e t h y l ketone with one of crocetindial (XXlV) and condensing the p r o d u c t CXVI with 2 moles of salt C X l V (cf. their echinenone synthesis, p. 317)"

O

(CXVl) (4) B y Isler et al. from fl-ionone, as outlined in 1956 and described in detail in 1959. fl-Ionone was converted into the retro-dehydro-Cxg-aldehyde (CXVlI) either as described in Helv., 1959, 42, 841 or, better, according to Scheme 21 (idem, ibid., 1959, 42, 847):

~

0

+

" / ~ ~ ~ C H ( O E t }2

EtMgBP_~

see p. 247.

/9- [onone

(XXl)

V~~cH(OEt)2

HE) ~_partiat redn.

~

0 (CXVII)

CXVll + BrMgC~CMgBr + CXVII

OH

I AcOH OAc

t

OAc

i OH|

diacetate )

~Oppenauer oxidn. 1535' - Dehydrocanthaxanthin Partia[ redn.; isomer'isn. Canthaxanthin (CXV) S c h e m e 2 I.

320

THE C A R O T E N O I D GROUP

7

In t h e technical synthesis of c a n t h a x a n t h i n (cf. Islet et al., Chimia, 1961, x5, 208) t h e d e h y d r o i s o z e a x a n t h i n diacetate is o b t a i n e d by t r e a t i n g synthetic I5,I5'-dehydro-/~-carotene (see p. 270) with N-bromosuccinimide in chloroformacetic acid (cf. p. 262) (for its use as a food-colouring m a t t e r , cf. Islet et al., loc. cir.). Astaxanthin. I n 1933, K u h n and Lederer (Ber., 1933, 66, 488) showed t h a t t h e green colouring m a t t e r of lobster eggs, ovoverdin, is a carotenoid-protein complex (or " c h r o m o p r o t e i n " ) which on d e n a t u r a t i o n with acetone yields a red c o m p o u n d a n d colourless protein. T h e red c o m p o u n d was n a m e d " o v o - e s t e r " since on t r e a t m e n t with alkali it was converted into a n o t h e r coloured substance, astacene (astacin). I n 1938, however, K u h n and IV. A. S6rensen (ibid., 1938, 7x, 1879) showed t h a t " o v o - e s t e r " was n o t an ester of astacene b u t was a new coloured substance, astaxanthin, which was converted into astacene w h e n exposed to air in the presence of alkali: on t r e a t i n g a pyridine solution of a s t a x a n t h i n with butanolic p o t a s s i u m h y d r o x i d e in t h e absence of air t h e deep blue enolate is formed which on exposure to oxygen r a p i d l y absorbs two moles of the gas with t h e f o r m a t i o n of astacene (red). Meanwhile, Karrer et al. (Helv., 1934, x7, 745; 1935, z8, 96) h a d shown t h a t astacene is C40H4sO 4 and gives a bisphenazine derivative with o-phenylenediamine (but only a dioxime) so indicating t h e presence of two p o t e n t i a l ~-diketone groupings. Oxidation of astacene and of its bisphenazine derivative with p e r m a n g a n a t e (which gave d i m e t h y l m a l o n i c and d i m e t h y l m a l o n i c + xx-dimethylsuccinic acids, respectively) led to t h e formulation of astacene as 3,4,3',4'-tetra-oxo-~-carotene. [Subsequently, I.R. and P.M.R. d a t a (f. B. Davis and Weedon, Proc. Chem. Soc., I96O, 182) indicated t h a t astacene exists, in solution, essentially in the di-enol form, shown]. I t followed t h a t a s t a x a n t h i n is a di-~-ketol, this p a r t i c u l a r s t r u c t u r e being favoured on a c c o u n t of t h e closeness of its absorption m a x i m u m to t h a t of astacene (Kuhn and S6rensen, 1938, loc. cir.):

OH

~D 0

0

~ ~v~ l

/~2s

0

OH

7

K E T O - AND H Y D R O X Y K E T O - C A R O T E N O I D S

321

A s t a x a n t h i n is typically an animal carotenoid being the major carotenoid of m o s t marine crustaceans and asteroids; it occurs in three forms - free, esterified (e.g., as dipalmitate), and combined with protein (as chromoproteins, which are variously blue, green, or brown and sometimes water-soluble). The dark blue colouring m a t t e r in the lobster's shell is an astaxanthin-protein complex ("o~crustacyanin"), which on t r e a t m e n t with acetone, or acid, or on heating (as during cooking), undergoes d e n a t u r a t i o n yielding a s t a x a n t h i n (red) and protein. a-Crustacyanin (M 35o,ooo; ~max.632 m/~, in water) contains ~ 3 % of its weight of a s t a x a n t h i n and can be reconstituted in vitro by treating a s t a x a n t h i n with the protein ("apocrustacyanin") obtained on d e n a t u r a t i o n of natural ~-crustacyanin with acetone. The means by which the a s t a x a n t h i n is bound to the protein is unknown b u t a p p a r e n t l y involves all four of its oxygen functions (R. K u h n and H. Ki~hn, Angew. Chem. internat. Edn., 1966, 5, 957; W. L. Lee and P. F. Zagalsky, Biochem. J., 1966, xox, 9c). A s t a x a n t h i n has also been found in bird feathers (cf. Fox, Comp. Biochem. Physiol., 1962, 6, I; V6lker, Fortschr. Chem. org. Naturstoffe, 196o, x8, 182) and locusts (Goodwin, Biochem. J., 1949, 45, 472) b u t only rarely in the vegetable kingdom (cf. Kuhn et al., Bet., 1939, 72, 1688; Tischer, Z. physiol. Chem., 1941, 267, 281). (Where a saponification step was included in the original isolation procedures, astacene rather t h a n a s t a x a n t h i n was obtained: it seems likely t h a t the colouring m a t t e r originally present was a s t a x a n t h i n in each case, the astacene being an artefact). For other carotenoids with an end group of the a s t a x a n t h i n t y p e (and consequent instability to base in the presence of oxygen), see flexixanthin (below) and p. 332. A staxanthin crystallises from aqueous pyridine in lustrous dark red plates, m.p. 2160 (decomp., uncorr.), Amax. 493 m/~ (pyridine) (with two subsidiary m a x i m a according to Kuhn and SOrensen, 1938, loc. cit.; one broad band in carbon disulphide according to Goodwin and Srisukh, Biochem. J., 1949, 45, 263); diacetate, m.p. 2o3-2o5~ dipalmitate, m.p. 72o (Kuhn and SOrensen, 1938,

loc. cit.). Astacene crystallises from aqueous pyridine in fine curved needles, m.p. 2280 (uncorr.), ~max. ~ 512 m/~ (carbon disulphide) (Kuhn et al., 1939, 1933, loc. cit.), or from chloroform-ethanol in deep-purple tapered rods, m.p. 232-233 ~ (uncoIT.) ; ~max. 498 m ~ (in pyridine, one broad band) (Davis and Weedon, loc. cit.; B.P., 917,241/1963); for I.R. and P.M.R. data, cf. Davis and Weedon, loc. cit. Astacene diacetate, has m.p. 235 ~ (decomp., uncorr.); dipalmitate, m.p. 121~ bisphenazine derivative, m.p. 231~ (uncorr., from chloroform-ethanol) (Kuhn etal., Z. physiol. Chem., 1933, 220, 229; 1939, loc. cit.; Davis and Weedon, loc. cit.). On partition between petrol and 9 o % methanol, astacene is found in the lower layer; on adding a little more water it is precipitated as a " s o a p " at the interface b u t remains in the lower layer if this is alkaline. Synthesis o/astacene (Davis and Weedon, loc. cir.). B y shaking a suspension of synthetic c a n t h a x a n t h i n (p. 320) in a mixture of benzene and potassium tert-butoxide in tert-butanol under oxygen, autoxidation x to the carbonyl groups occurs with the formation ( ~ 4o%) of astacene:

322

THE CAROTENOID GROUP

I

7

I

By using I5,I5'-dehydrocan~axanthin (p. 319), wMch is more soluble, the reaction proceeds more rapidly (2"5 hr.) and gives a high yield (93%) of I5,x5'dehydro~t~,e~, m.p. 197-198~ which can be converted into ast~cene in the usual way (p. 247). Synthesis o/~taxanthin (A. P. Le/twick and Weed~, Chem. Comm., I967, 49) - From synthetic astacene by reducing with borohydnde to give the te~aol corresponding to the te~aketo- form of astacene followed by oxidation of the allylic hydroxyl groups with 2,3-dichloro-5,6-dicyanobenzoquinone. Flexixanthin, with one end group of the astaxanthin type and the other acyclic, has been isolated from a Flexibacter sp. (an aquatic micro-organism); on treatment with base in the presence of oxygen it yields the corresponding dehydro-compound with an astacene-type end group. It is the 3-hydroxy derivative of deoxy-flexixanthin, p. 318 (which see for ref.). Capsanthin and capsorubin. Capsanthin and, in much smaller amount, capsorubin occur together in the ripe fruits of Capsicum annuum (red peppers) (Zechmeister and Cholnohy, Ann., 1934, 509, 269; I935, 516, 3o; cf. also, Cholnoky et al., Acta Chim. Acad. Sci. Hung., 1955, 6, 143). Spectral data and the isolation of /~-citraurin (3-hydroxy-/~-apo-8'-carotenal: p. 312) on treating capsanthin (apparently C40HssO3) with hot alkali (reverse aldol-condensation) established the part structure CXVlII, where " R " contains a second hydroxyl group (Zechmeister and Cholnohy, Ann., I937, 53o, 291): R

These authors showed t h a t capsorubin, apparently C40I-~O 4, has two hydroxyl groups and (probably) a nonaenedione chromophore, and suggested t h a t the two end-groups are identical (and apparently acyclic, by analysis) and the same as t h a t (R) in capsanthin. Warren and Weedon (J. chem. Soc., 1958, 3972) confirmed t h a t capsorubin has a nonaenedione chromophore, showed t h a t it yields crocetindial (XXlV, p. 248 ) on treatment with base (cf. capsanthin, above), and eliminated, by synthesis, four of the structures for the substance proposed earlier. Meanwhile, Cholnohy et al. (Ann., I957, 606, 194) found t h a t samples of capsanthin and capsorubin dried in vacuo at a considerably higher temperature than normally used for polyenes analysed for C40Hs6Os and C40H5604, respectively, indicating t h a t both end-groups in the two compounds were cyclic. The further elucidation of the structure of these compounds is a good illustration of the application of modern methods to carotenoid chemistry.

7

KETO- AND HYDROXYKETO-CAROTENOIDS

~2~

In I96O, Weedon et al. (Proc. chem. Soc., I96O, 19) and Entschel and Karrer (Helv., I96O, 43, 89) discovered independently t h a t Oppenauer oxidation of capsanthin gave a hydroxy-diketone ("capsanthone") (the cyclohexenyl ring OH resists oxidation: cf. Weedon et al., J. chem. Soc., 1961, 4oi9), which showed a carbonyl band in the infrared characteristic of a cyclopentanone; Entschel and Karrer showed further t h a t capsorubin similarly gives a tetraketone, "capsorubone", containing two C~ ring keto-groups. Weedon's group found also t h a t the high-field (3 > 8.5) portion of the P.M.R. spectrum of capsanthin showed, in addition to methyl bands due to the known end-group (cf. CXVIII), three CH 3 singlets characteristic of methyl groups attached to fully-substituted saturated carbon; capsorubin showed these three singlets only, but with twice the intensity, strongly supporting the earlier suggestion t h a t the "unknown" end-group of capsanthin and the two end-groups of capsorubin are all the same. In capsanthone, the three bands ascribed to the C 5 ring methyls occurred at consistently lower field than the corresponding bands in synthetic CXIX, indicating t h a t the C5 ring methyls in capsanthone are uniformly deshielded by the o

ring keto-group, which therefore was placed at Cos) so establishing the position of the OH group in the original carotenoids (substantiated later by a study of the spectra of model compounds: Weedon et al., I961, loc. cir.): GH

140

% H

Capsorubin

[Entschel and Karmr (loc. cir.) proposed the same structures, b u t essentially on biogenetic grounds]. Confirmation of these structures was provided by ozonising the diacetates of capsanthin and capsorubin: one of the products formed on t r e a t m e n t of either ozonide with peroxide was 1,2,2-trimethyl-4-hydroxycyclopentane-I-carboxylic ((39) acid (H. Faigle and Karrer, Helv., I96I, 44, I257; cf. Cholnoky and Szabolcs, Experientia, I96O, x6, 483).

324

THE CAROTENOID

7

GROUP

The full stereochemistry of capsorubin has since been deduced, as follows: (a) R. D. G. Cooper, L. M. Jackman and Weedon (Proc. chem. Soc., 1962, 215) synthesised C X X (as a DL mixture), reduced it with borohydride to give a mixture of the cis- (CXXI and enantiomer) and trans- (CXXlI and enantiomer) hydroxy-acids which were separated, and identified, by the ability of the cis compounds to undergo lactone formation. T r e a t m e n t with methyl-lithium gave the corresponding m e t h y l ketones which were condensed with crocetindial (XXlV, p. 248 ) to give C X X l I I (plus its enantiomer and the meso-form) and 1_CO,?.H

\

CO2H

0

0

/

CO2t"1

C_O2H

OH

OH

v (CXX)

(CXXI)

(CXXII)

C X X I V (with its enantiomer and meso-form), respectively, - and it was the latter which showed the same spectral (P.M.R.) and chromatographic properties as natural capsorubin. In confirmation, Karrer's group showed later (v/a a t t e m p t ed lactone formation and P.M.R. spectra) t h a t the oxygen functions in the C9 acid obtained (above) as a degradation product of both capsanthin and capsorubin are disposed trans to one another (Helv., 1964, 47, 741) 9 (b) The absolute configuration at C(5 ) in capsorubin (and similarly in capsanthin) is now known to be the same as t h a t at C(1) of ( + ) - c a m p h o r [by relating the C9 acid mentioned above to ( + ) - c a m p h o r through a common degradation product, camphoronic acid, HO,C.CMe2-CMe(CO2H).CH2.CO,H: Faigle and Karrer, Helv., 1961, 44, 19o4] so t h a t in natural capsorubin the end groups have the stereochemistry shown in C X X l V . HO

M~ Me ....

0 ~.~/,.,,~. Me - M ~ ......~ o ~

H

(CXXIII)

H

H

Me M~

M O ~ Me ..... '~

Me . . . . . . ~..~./m..~

OH

Me M~

(CXX IV)

Synthesis: of (• as above. Physical properties. Capsanthin has m.p. 175-176o (corr., from carbon disulphide or petrol), 1760 (Kofler block), ~max. 498, 469 (hexane) ; 476 m # (ethanol); [x]Cd + 3 6 ~ (chloroform) (Zechmeister, Cholnoky et al., Ann., 1934, 5o9, 269; 1957, 6o6, 194; J. Amer. chem. Soc., 1944, 66, 186) ; a series of di-esters, including the diacetate, m.p. 15 o~ and dipalmitate, m.p. 95 ~ has been prepared. Capsorubin has m.p. 218 o (Kofler block, from carbon disulphide), ~max. 502, 470, 441 (hexane); 541, 502, 467 m/~ (carbon disulphide); [a ]Hg-C O0 (Cholnoky

7

KETO- AND HYDROXYKETO-CAROTENOIDS

325

et al., loc. cir.; Ann., 194 o, 543, 248); a series of di-esters, including the diacetate, m.p. I8O ~ and dipalmitate, m.p. 85 ~ has been prepared. Capsanthin and capsorubin are found only rarely, apart from in red peppers: capsanthin occurs in the anthers of Lilium tigrinum (with antheraxanthin, its probable precursor, cf. p. 345: Karrer and A. Oswald, Helv., 1935, 18, 13o 3) and probably in other Lilium spp. (A. Seybotd, C.A., 1954, 48, 13834; B. G. Savinov and S. E. tfudritskaya, C.A., 196o, 54, 3614); for other sources of capsorubin, see Karrer and S. Ramasvamy, Helv., 1951, 34, 2159; Savinov and Kudritskaya, loc. cir. Regarding their biogenesis, cf. p. 345. Kryptocapsin, m.p. 16o-161 ~ Amax. (52o), 486 m/~ (benzene), a minor component of the carotenoid mixture in red peppers (cf. above), was first described by Cholnoky et al. (Acta Chim. Acad. Sci. Hung., 1955, 6, 143; 1958, x6, 227). Its structure followed (Cholnoky, Weedon et al., Tetrahedron Letters, 1963, No. 19, 1257) from its Oppenauer oxidation to a cyclopentanone ("kryptocapsone", oH

o

'max. 1739 cm.-~), chromic acid oxidation to camphoronic acid (HO~C.CMe~. CMe(CO~H).CHs.CO~H), degradation by alkali to ~-apo-8'-carotenal (cf. capsanthin), and a comparison of the P.M.R. spectra of kryptocapsin and kryptocapsone with those of capsanthin and capsanthone. The optically inactive colouring matter, m.p. I3i-i320, synthesised by the base-catalysed condensation of synthetic ~-apo-8'-carotenal (CXXV: cf. p. 247) and CXXVI (and enantiomer: from CXXII, above), had the same spectral and chromatographic properties as natural kryptocapsin. OH

(cxxv) (cxxvl)

! (e) Carotenoid carboxylic acids

Torularhodin, m.p. 210-2120 (uncorr., from toluene), ~max. 535, 507 (petrol), 582, 541, 502 m/~ (carbon disulphide), occurs (along with structurally-related torulene, p. 28o) in the red yeast Torula rubra (E. Lederer, Compt. rend., 1933, 197, 1694; cf. Karrer and J. Rutschmann, Helv., 1943, 26, 21o9; 1945, 28, 795; 1946, 29, 355). In 1959, Isler et al. (ibid., 1959, 42, 864) obtained a compound having the following structure by total synthesis and showed t h a t it was identical with natural torularhodin :

326

THE CAROTENOID GROUP

7

J

H

Torutarhodin

It gives a methyl ester, m.p. 172-173 ~ with diazomethane which shows weak vitamin A activity. Regarding the mode of biosynthesis of torularhodin, cf. P. 345Synthesis. Islet et al. (loc. cit.) obtained the methyl ester (m.p. 176-1770) by a Wittig reaction between the ylide, P h 3 P = C M e . CO2Me, and the 15,I 5'-dehydrofl-C3~-aldehyde (cf. p. 247) followed by partial reduction; alkaline hydrolysis gave torularhodin. Neurosporaxanthin (C35), first detected by F. Haxo in Neurospora crassa and since found in N. sitophila and certain other fungi, has m.p. I92 ~ ~max. (504), 477 m/, (petrol) (Haxo, Arch. Biochem., 1949, 20, 400; M. Zalokar, Arch. Biochem. Biophys., 1957, 7o, 568; L. R. G. Valadon and R. C. Cooke, Phytochem., 1963, 2, Io3;Aasen and Jensen, Acta Chem. Scand., 1965, 19, 1843). Its structure (Aasen and Jensen, loc. cir.) was inferred from an inspection of its visible and I.R. absorption spectra and confirmed by direct comparisons [spectra, mixed chromatograms, partition behaviour (p. 245)] of the natural acid, the methyl ester prepared from it (diazomethane), and the polyene alcohol prepared from the ester by reduction with hydride, with synthetic samples of these three compounds (cf. below). /C%H

Neucosporaxanthin

Synthesis: Isler et al. had synthesised both the methyl ester (m.p. 143 ~ corresponding to neurosporaxanthin and the acid itself in 1959 using a route analogous to that used for torularhodin, above, but starting from the corresponding C3, aldehyde (p. 247). Biogenesis- probably via oxidative degradation of torulene or ),-carotene, with which it co-occurs in nature: cf. torularhodin. Bixin (C~5), the colouring matter of the plant Bixa orellana, was first isolated in the crystalline state by C. Etti (Bet., 1878, 11,864). Kuhn and his collaborators showed t h a t oxidation of bixin with permanganate or chromic acid gave acetic acid corresponding to four side-chain methyls, elucidated the structures of the fragments obtained on ozonolysis, and, in 1932 (ibid., 65, 646) proposed structure C X X V I I a for the compound. Confirmation of this was provided by the investigations of Karrer et al. (Helv., 1932, 15, 1399) which included the identification of perhydronorbixin (cf. below) prepared from natural bixin with a sample obtained by total synthesis:

7

CARBOXYLIC ACIDS ,A

s

k (CXXVll)

A

A

A

327 A

.c

R

a;R- Me(.bixin) b ; R ,, H ( ,, nof'bix in )

I t was in the bixin series t h a t cis-trans isomerism was first encountered in the carotenoid field. N a t u r a l bixin ("labile bixin"), m.p. I98 ~ (corr., rapid heating, Kuhn et al., ibid., I928, IX, 716) ~max. 523, 489, 457 m/~ (carbon disulphide), is a cis c o m p o u n d which on t r e a t m e n t with iodine gives (cf. Karrer et al., ibid., I929, I2, 74 I) the all-trans form, "stable" (or "fl") bixin, m.p. 216-217 ~ (decomp.), ;t,nax. 526, 49I, 457 m/~ (carbon disulphide). Jackman, Weedon, and co-workers (J. chem. Soc., 1961, 1625) have shown, from an analysis of the P.M.R. spectra of bixin and related compounds, t h a t n a t u r a l bixin has the cis-I6-structure. Saponification of the "labile" and " s t a b l e " forms of bixin gives the corresponding diacids, the norbixins, m.p.'s 254-255 o and > 3oo ~ respectively, whilst t r e a t m e n t with d i a z o m e t h a n e gives the dimethyl esters, methyl natural bixin, m.p. 164 ~ and methyl "stable" bixin, m.p. 2Ol ~ respectively (Karrer et al., loc. cit.). The corresponding dialdehyde, bixindial, which can be converted into norbixin (p. 267), is of i m p o r t a n c e in t h a t it is one of the products obtained on oxidation (p. 259) of lycopene and its derivatives (e.g. spirilloxanthin, p. 308) with permanganate. Syntheses (of all-trans-methylbixin). (i) B y R. Ahmad and Weedon (J. chem. Soc., 1953, 3286) 9 "~; PhLi CH~ =CMe. CH2C1 > HOCHg. CMe =CH. C = C H > ROCHg. CMe=CH. C----CLi c, diketone; (el. p. 246)H (B + (ROCHs" CMe=CH" C~C" CMe-----CH"CH--)" H(B. inO,; CHt(COtH)t_~. (HO~C. CH =CH. CMe =CH. C~C. CMe =CH 9CH =)3 CHtNt; Ht

-+ Methylbixin

(2) Later, b u t more conveniently, b y a W i t t i g reaction between 2 moles of P h 3 P = C H . C O ~ M e and crocetindial ( X X l V p . 248 ) (Isler et al., Helv., 1957, 4o, 1242: also diethyl and dicetyl esters) or alternatively, between 2 moles of the ylide, P h 3 P = C M e . C H = C H . C O I M e and Isler's Cx4-dialdehyde (the centraldihydro-derivative of XXVlI, p. 248 ) (E. Buchta and F. Andree, Bet., 1959, 92, 3111). (3) B y condensing 2 moles of MeO2C.CH~.PO(OEt)g (from m e t h y l chloroacetate and triethylphosphite) with crocetindial (H. Pommer, Angew. Chem., 196o, 72, 911). Crocetin (Cs0), as its digentiobiose ester, crocin, is responsible for the colour of saffron (obtained from the stigmas of a u t u m n crocus). Karrer et al. (Helv., 1928, xx, 513, 711; 1929, x2, 985; 1932, x5, 1399 ) and K u h n and L'Orsa (Ber., 1931, 64, 1732) showed t h a t crocetin is a dicarboxylic acid with seven double bonds and four side-chain methyls. Karrer (1932, loc. cir.) then proposed the following

328

THE CAROTENOID

GROUP

7

structure and subsequently confirmed this by synthesising (Helv., 1933, x6, 297) the perhydro-derivative and identifying it with perhydrocrocetin. Perhydronorbixin (above) has been related to perhydrocrocetin by stepwise degradation (H. Raudnitz and J. Peschel, Ber., 1933, 66, 9Ol):

HO2r

~

~

~

~

~

Crocetin

~

I

/ C0 2H

I

P a r t of the crocetin in crocin is present as a cis-isomer which gives a dimethyl ester, m.p. 141% convertible by iodine or heat into the all-trans form, m.p. 222 o (tfuhn and Winterstein, ibid., 1933, 66, 2o9); aU-trans ("stable") crocetin has m.p. 285 o and tmax. 482, 453, 426 m/~ (carbon disulphide). Synthesis o/ crocetin diesters. Originally by H. H. Inho~en et al. (Ann., 1953, 580, 7), and subsequently by Islet et al. (Helv., 1957, 4o, I242), Buchta and Andree (Bet., 196o, 93, I349), and Pommer (Angew. Chem., 1960, 72, 911) using routes essentially similar to those used for methylbixin, above. Azafrin (Cz~), m.p. 2120 (corr.), tmax. 458,428 m # (chloroform), is the colouring m a t t e r of the roots of the South American plant, Escobedia scabri/olia; it gives a methyl ester, m.p. 193 ~ Extensive degradative work by Kuhn and co-workers (Bet., 1931, 64, 333; 1933, 66, 883; 1934, 67, 885; Ann., 1935, 5x6, 95), including the preparation of a derivative ("anhydroazafrinone amide") also obtained by stepwise degradation of/~-carotene, led to the structure: co~ OH

Azafrin

The hydroxyl groups are probably in the trans-diaxial configuration (no intramolecular hydrogen-bonding effects in the infrared; the bulky polyene chain is presumed to be equatorial: H. Mi~ller and 1farter, Helv., 1965, 48, 291).

( f ) Allenic carotenoids Fucoxanthin, the principal carotenoid of the brown algae (Phaeophyceae - e.g. seaweeds), is also found in diatoms, and is probably the most abundant xanthophyll in nature; it has m.p. 166-167 o (red leaflets from ether-petrol), A1nax. 478, 449, (425) (petrol); 5o8, 478 m p (carbon disulphide); it is rapidly decomposed by alkali (Heilbron and R. F. Phipers, Biochem. J., 1935, 29, 1369; A. Jensen, Acta Chem. Scand., 1961, x5, 16o 4, 16o5; F. G. Torto and Weedon, Chem. and Ind., 1955, 1219). Work with this compound has been hampered by its instability to base and acid and its tendency to undergo marked deterioration during isolation (frequently from the seaweed Fucus vesiculosus). Oxidation with permangan-

7

ALLENIC CAROTENOIDS

32 9

ate (as on p. 2431 and examination of the I.R. spectrum revealed the presence of a Me, C~/cH,__ C residue, and of an allene grouping (for the first time in a carotenoid), conjugated keto, and isolated keto (or ester) groups; analysis suggested the formula C40H56_e00e. Subsequent studies by Weedon et al. (Proc. chem. Soc., 1964, 419) and A . Jensen (1961, loc. cir.; Acta Chem. Scand., 1964, z8, 84o, 2oo5) demonstrated the presence of an acetate function (alkaline hydrolysis, of both fucoxanthin and its perhydro-derivative); of one secondary or primary and one tertiary hydroxyl group (under acetylating conditions a monoacetate was produced, and this readily underwent dehydration with phosphorus oxychloride); and established the molecular formula as C,2H5806 (mass spectrometry). In addition, Weedon's group succeeded in isolating a series of polyene aldehydes from the mild oxidative degradation (zinc permanganate) of fucoxanthin, determined the structures of three of these by physical and degradative methods, and tentatively formulated fucoxanthin as below, a structure which was consistent (a) with the observed optical activity (Antia, Canad. J. Chem., 1965, 43, 3o2; Weedon et al., loc. cir.); and (b) with Jensen's finding (1964) that following ozonolysis of fucoxanthin monobenzoate a fragment (C15?) can be isolated, which contains allene, acetate, and tertiary OH functions but lacks the benzoate group (derived from the secondary OH group in fucoxanthin, which must therefore be at the "other end" of the molecule):

HO

~OAc OH

Fucoxanthin

This was later confirmed by Weedon and co-workers (Chem. Comm., 1966, 515) who elucidated the structures (spectra; molecular formulae by mass spectrometry) of three allenic fragments Ecleavage at C(1~), C(u'), and C0, ), respectively] produced by mild oxidation (see above), and carried out a series of interconversions (which the allene function generally survived) elucidating the structures of the various compounds formed as above. Three noteworthy reactions of fucoxanthin are: (a) Reduction with lithium aluminium hydride followed by treatment with dilute chloroformic hydrogen chloride (removal of aUylic hydroxyl, and epoxide rearrangement: cf. p. 293) gives a mixture of (epimeric) furanoid oxides with the following structure (cf. also foliachrome p. 330):

140 .

0

__

j

OH

(b) On dehydration of fucoxanthin acetate [OAc at CO)]-cf. a b o v e - a new double bond is produced at C(4,)--C(5,), without further rearrangement. (c) On leaving overnight adsorbed to a weakly alkaline (alumina or magnesia)

330

THE

CAROTENOID

7

GROUP

column, fucoxanthin undergoes r e a r r a n g e m e n t and d e c o m p o s i t i o n - the major p r o d u c t of which, isolucoxanthin, has the structure shown and is p r e s u m a b l y formed as follows ( Weedon et al., 1966, loc. cit." A. Jensen, Acta Chem. Scand., I966, 20, I728)"

Fucoxanthin

HOO/~. ~ %~OAr OH

OH

Isofucoxonthin

Those fucoxanthin derivatives having intact epoxy and keto groups react similarly. Foliaxanthin, a carotenoid first isolated from paprika b y L. Cholnoky in 1955 and later reported to occur in various leaves (Cholnoky et al., Chem. Comm., 1966, 4o4), has m.p. I28 ~ ~max. (EtOH) 467, 439, 416 m/z, and contains (spectra) a nonaene chromophore and OH and C = C = C (Vmax. 192o cm. -1) functions. I t gives a diacetate on mild acetylation and with chloroformic hydrogen chloride gives a furanoid derivative ( ;tmax. shift ~ 18 m/z: cf. p. 293),/oliachrome, m.p. 148~ The molecular formulae and n u m b e r of O H groups in each compound were established b y mass s p e c t r o m e t r y (cf. p. 257). Foliachrome is a p p a r e n t l y identical (comparison of spectra, chromatographic properties, mass spectra) with the m i x t u r e of epimeric furanoid oxides derived from fucoxanthin, above (Weedon et al., 1966, loc. cit.) :

HO Foliaxantbin

" ~OH OH

Neoxanthin, widely distributed in nature (p 238), has long been known to contain an epoxide function (effect of H e : p. 293) and (probably) a nonaene chromophore; the partition behaviour (p. 245) of n e o x a n t h i n and of the compounds produced on treating it with acid in m e t h a n o l (dehydration of t e r t i a r y h y d r o x y l ; m e t h y l a t i o n of allylic hydroxyl) and in acetone (dehydration only) indicated the presence of two secondary and one t e r t i a r y h y d r o x y l groups (cf. Krinsky et al., Biochemistry, 1966, 5, 1814; Curl, J. Food Sci., 1965, 3o, 426); coupled with other d a t a this led to a t e n t a t i v e formulation for n e o x a n t h i n (as the 6',7'-dihydro-derivative of the formula shown above) b u t this was later disproved by C. O. Chichester and co-workers (Chem. Comm., 1966, 807; 1967, 484) who showed t h a t n e o x a n t h i n (from spinach leaves) contains an allene function and t h a t it is identical with foliaxanthin.

(g) Carotenoids of uncertain constitution Below are m e n t i o n e d some of the m a n y carotenoids which have been reported whose structures are uncertain or entirely unknown.

7

CAROTENOIDS OF UNCERTAIN CONSTITUTION

331

Zeinoxanthin, m.p. 175-176~ Amax. 474, 445, 422 m/~ (hexane), [aiD + 5 ~176 (acetone), isolated from corn by E. N. Petzold and F. W. Quackenbush (Arch. Biochem. Biophys., 196o, 86, 163), shows no vitamin A activity and has spectral and chromatographic properties which suggest t h a t it is 3-hydroxy-a-carotene. A compound frequently detected in fruits has also been tentatively assigned this structure (A. L. Curl, Food P,es., 1956, 21, 689):

Ho

Myxoxanthophyll, from Oscillatoria rubescens (Heilbron and B. Lythgoe, J. chem. Soc., 1936, 1376; Karrer and Rutschmann, Helv., 1944, 27, 1691) and probably present in other blue-green algae (Goodwin, J. gen. Microbiol., 1957, 17, 467), has m.p. 182 ~ ;tmax. 5o3, 47 x, 445 m/~ (ethanol) ; it analyses for C40H560~, gives a tetra-acetate which contains two free (tertiary) hydroxyl groups, and (probably) contains one isolated carbonyl group; J. Tischer's aphanizophyll (from algae: cf. Z. physiol. Chem., 1944, 281,143 ) has similar irma,, values, but is more polar (Hertzberg and Jensen, Phytochem., 1966, 5, 565). O. rubescens also contains oscillaxanthin, an acidic carotenoid with ~max. 568, 528, 494 m/~ (carbon disulphide) (Karrer and Rutschmann, loc. cir.). Phlei-xanthophyU, from Mycobacterium phlei Vera, has the s a m e ~max. values as myxoxanthophyll but is even more polar* (Hertzberg and Jensen, Acta Chem. Scand., 1966, 2o, 1187). Pectenoxanthin (C40H5~_5603) and pentaxanthin (C40H54_5805), both from the marine world, have m.p.'s 182 o and 21o 0 and22max. (carbon disulphide) 518, 488, 454 and 5 o6, 474, 444 ml, (E. Lederer, Compt. rend. Soc. biol., 1934, II6, i5o; Bull. Soc. chim. biol., 1938, 2o, 611). Peridinin, found in various Peridinium species [Dinophyceae (algae)], is very polar, is, like fucoxanthin, decomposed by alkali, and has 2max. 516, 483 m/~ (carbon disulphide) (cf. Strain et al., Biol. Bull., 1944, 86, 169; Zechmeister et al., Arch. Biochem. Biophys., 1953, 44, 189); it is probably identical with sulcatoxanthin, probably C40H5,O 8, found in a sea anemone (or the algae therein?) by Heilbron et al. (Biochem. J., 1935, 29, 1384). Siphonoxanthin, a polyhydroxyketo-carotenoid with 2max. 480, 452, (428) mp (petrol), is the major xanthophyll in members of the order Siphonales (algae) occurring partly free and partly esterified (siphonein) (Strain, 1958: full ref., p. 238). Sarcinaxanthin, m.p. 149I5 o~ 2max. 469, 44o, (415) m/, (petrol), hypophasic, and sarcinene (epiphasic) occur in Sarcina lutea (a bacterium: cf. Takeda and Ohta, Z. physiol. Chem., I94I, 268, I). Renieraxanthin (C40H5oO,?), m.p. I6I ~ contains a conjugated carbonyl group; it accompanies the aromatic carotenoids in Reniera japonica (cf. p. 277; Yamaguchi, 1957). * The high polarity of this compound (and possibly certain other highly oxygenated carotenoids mentioned in this section) is due to its occurring in nature linked to a sugar residue: S. L. Jensen, 1967.

332

THE CAROTENOID GROUP

7

Aleurixanthin, from the fungus Aleuria aurantia, is a monohydroxy derivative of, perhaps, 7-carotene (Jensen, Phytochem., I965, 4, 925)" Diaflinoxanthin, from diatoms (cf. Strain et al., I944, 1958, loc. cir.), is probably a diol epoxide (cf. Krinsky, Anal. Biochem., 1963, 6, 293). Diatoxanthin (polarity and chromophore similar to, but distinct from, those of zeaxanthin, p. 297)* and dinoxanthin have been isolated from various algae (Strain et al., I944, I958, loc. cir.; Jeffrey, Biochem. J., I961, 80, 336; Allen et al., J. gen. Microbiol., I964, 34, 2.59; 196o, 23, 93). Hydroxyechinenone (from Euglena gracilis: Krinsky and Goldsmith, Arch. Biochem. Biophys., 196o, 9I, 27I), adonirubin (from Adonis annua: K. Egger, Phytochem., 1965, 4, 6o9), and adonixanthin (same source) all show astaxanthin-like instability towards alkali in the presence of oxygen (cf. p. 32o) and all three are considered to have one end group of the astaxanthin type (the other end group being formulated as a r-carotene or oxy-fl-carotene type); A. annua also contains a carotenoid similar to the colouring matter in Euglena and now identified as 3'-hydroxyechinenone (Egger, loc. cit.; Le[twick and Weedon, Chem. Comm., 1967, 49). Okenone, m.p. 152.5 o (violet rods from acetone-petrol), ~max. 516, 485, (460) mp (petrol), is the major carotenoid of the purple bacterium Chromatium okenii; it contains aryl, conjugated keto (comparison of visible spectra in petrol and ethanol; I.R.) and methoxyl (I.R.) functions. Warmingone, the major carotenoid of C. warmingii, apparently contains conjugated keto and hydroxyl functions and has a similar skeleton to that of rhodopin, p. 307 (Schmidt, Jensen and Schlegel, Arch. Mikrobiol., 1963, 46, 117, 138; 1965, 52, 132). Reticulataxanthin and (in small amount) tangeraxanthin occur in tangerine (Citrus reticulata) peel; both contain hydroxyl groups and have chromophores of (probably) ten and eleven double bonds, respectively, terminated by a carbonyl group (Curl, J. Food Sci., 1962, 27, 537). Reticulataxanthin and the recently discovered citrus carotenoids citranaxanthin (C33) and sintaxanthin (C3t) have all been assigned structures of the type: cyclic end group-polyene chainCO-CH 3 (Yokoyama et al., J. org. Chem., 1965, 3o, 2481, 2482, 3994). The following carotenoids are all known to be monoepoxides (from the characteristic spectral effect on adding acid: cf. p. 293). Trollixanthin, m.p. I55-I56~ ~max. 471, 449, 422 mp (hexane), from the petals of Trollius europaeus, is probably (Karrer et al., Helv., 1955, 38,638) 6'-hydroxylutein-5,6-epoxide; the corresponding furanoid oxide, trollichrome, has m.p. 206 ~ The same flowers yield trolliflor (probably C40HseOs), m.p. 2oo ~ which has an acyclic nonaene chromophore" the furanoid form, trolliflavin, has m.p. 186-187 o (Lippert and Karrer, Helv., 1956, 39, 698). Taraxanthin, m.p. I84 ~ isolated from dandelion petals by K u h n and Lederer (Z. physiol. Chem., 1931, 2oo, lO8; cf. also Booth, Phytochem., 1964, 3, 229) and since found (occasionally as low melting cis-forms) in the flowers of other plants (Eugster and Karrer, Helv., 1957, 4o, 69), is a triol epoxide with an absorption curve as for violaxanthin; the furanoid form, tarachrome, had m.p. 162-168 ~ * Probably 7,8-dehydrozeaxanthin (A. K. Mallams et al., Chem. Comm., 1967, 3oi).

8

BIOSYNTHESIS

OF H Y D R O C A R B O N S

333

Valenciaxanthin and sinensiaxanthin (in Valencia oranges) and persicaxanthin (in peaches) are notable for the shortness of their chromophores (six, seven, and six double bonds, respectively) (Curl et al., J. agric. Food Chem., 1954, 2, 685; Food Res., 1959, 24, 413). 8. The biosynthesis of carotenoids

(a) Biosynthesis of hydrocarbons (i) General The early observation that the tomato and some other fruits fail to produce carotenoids if ripened in the absence of oxygen* suggested that carotenoids are formed from more saturated precursors by dehydrogenation (L. Zechmeister, "Carotinoide", Springer, Berlin, 1934; cf. Zechmeister et al., J. Amer. chem. Soc., 1946, 68, 197; Arch. Biochem., 1946, io, 113). In 1944-1946 two such compounds were discovered. In 1944, Zechmeister and A. Polgar (Science, 1944, ioo, 317) found that the carotenoids present in tomatoes and carrots were accompanied by a colourless compound, which was subsequently named phytofluene (Zechmeister and A. Sandoval, Arch. Biochem., 1945, 8, 425) and was shown to be an acyclic C40 isoprenoid hydrocarbon but to contain fewer double bonds than the true carotenoids and a shorter (pentaene) chromophore (idem, J. Amer. chem. Soc., 1946, 68, 197; V. Wallace and J. W. Porter, Arch. Biochem. Biophys., 1952, 36, 468). Phytofluene is a viscous oil and is very sensitive to aerial oxidation; it emits a characteristic intense greenish fluorescence on exposure to ultraviolet light. It frequently accompanies carotenoids in nature (though often in only trace amounts) (Zechmeister et al., 1945, loc. cit.; Arch. Biochem. Biophys., 1953, 47, 16o; cf. also below). Porter and F. P. Zscheile (Arch. Biochem., 1946, io, 547) found that tomatoes also contain a compound with a triene chromophore, later named phytoene (Porter and R. E. Lincoln, ibid., 295o, 27, 390), which was also sensitive to aerial oxidation but only very weakly fluorescent. It has since been found widely dispersed in nature (W. J. Rabourn and F. W. Quackenbush, Arch. Biochem. Biophys., 1953, 44, 159; cf. also below). A convenient source of phytofluene and phytoene, and also of ~-carotene, is carrot oil (Porter et al., ibid., 1952, 36, 468" 1954, 48, 267" Zechmeister and B. K. Koe, J. Amer. chem. Soc., 1954, 76, 2923). During the I94O'S, Porter and his collaborators had developed various strains of tomato which contained large amounts of one or more of those polyenes [including phytoene, phytofluene, ~-calotene (with a heptaene * The same effect has since been observed in c e r t a i n micro-organisms (M. Zalokar, Arch. Biochem. Biophys., 1954, 50, 71; M. M. Mathews, Photochem. Photobiol., 1963,

2, I).

334

THE CAROTENOID GROUP

7

chromophore" p. 275), and neurosporene (a nonaene, then known as "tetrahydrolycopene"" see p.276)] normally found therein only in traces. From an examination of the relative amounts of each of these hydrocarbons in related strains, Porter and Lincoln (I95O , loc. cit.) suggested that in the tomato, lycopene, the major carotenoid, is formed from phytoene by sequential dehydrogenation" phytoene (with three conjugated double bonds) --~ phytofluene (with five) > ~-carotene (with seven) --~ neurosporene (with nine) > lycopene (with eleven). Since then much (mainly circumstantial) evidence has accumulated which supports the theory and which also suggests that lycopene in other tissues is formed in the same way. (a) The close structural resemblance of phytoene, phytofluene, ~-carotene, and neurosporene with lycopene, inferred by Porter and Lincoln from the early work on these four compounds, was subsequently confirmed by Zechmeister and Koe (loc. cir.), who showed that each member of the PorterLincoln series (phytoene to lycopene) could be converted into the next by treatment with N-bromosuccinimide (this simultaneously provided an in vitro analogy for the proposed biosynthetic pathway). (b) It is now known that the carotenoids in a wide variety of natural (vegetable) sources are accompanied by phytoene, phytofluene, ~-carotene, and, less frequently, neurosporene IT. W. Coodwin, Biochem. J., 1952 , 50, 55o" 1953, 53, 538" M. Zalokar, Arch. Biochem. Biophys., 1954, 5o, 7 I (fungi); Porter et al., ibid., 1953, 46, 252" 196o, 88, 68 (tomatoes)" Goodwin, Biochem. J., 1952 , 51,458" 1956, 62, 346" A. L. Curl, J. agric. Food Chem., 1956, 4, x56" 1962, Io, 5o4; Food Research, 1959, 24, 413 (other fruits)" Porter et al., Arch. Biochem. Biophys., 1952 , 36, 468" I954, 48, 267 (carrots) ; A. E. Joyce, J. Sci. Food Ag., 1959, I o, 342" cf. also D. M. Eny, Arch. Biochem. Biophys., 1953, 46, 18; H. E. Wright et al., ibid., 1959, 8z, lO 7 (leaves) ]. (c) The addition of a trace of diphenylamine to a carotenoid-producing micro-organism arrests the formation of the true carotenoids but not of the more saturated polyenes (phytoene, etc.), which accumulate (G. Turian, Helv., 195o , 33, 1988; cf. Goodwin, Adv. Enzymol., I959, 21,320). S. L. Jensen et al. (Biochim. Biophys. Acta, 1958 , 29, 477) found that if the bacterium Rhodospirillum rubrum was so treated and then washed free of diphenylamine and suspended in a medium unable to provide carbon (for fresh "acetate" synthesis), the more saturated polyenes which had accumulated disappeared and were replaced by an equivalent amount of lycopene and its derivatives. (d) H. Claes (Z. Naturforsch., 1954, 9b, 461" 1956, x lb, 260" cf. also ibid., 1957, I2b, 4Ol) found that three mutants of the alga Chlorella vulgaris synthesised" phytoene only" phytoene, phytofluene and ~-carotene; and

8

BIOSYNTHESIS

335

OF H Y D R O C A R B O N S

phytoene, phytofluene, ~-carotene, neurosporene and l y c o p e n e , - the inference being that calotenoid biosynthesis was occurring along the Porter-Lincoln pathway and was being blocked at a different stage in each of the mutants. (For a parallel finding with a set of maize mutants, cf. Goodwin et al., Phytochem., 1966, 5, 581). (e) D. A. Beeler and Porter (Arch. Biochem. Biophys., 1963, ioo, 167) found that the extent to which 14C was incorporated into the polyenes produced by a tomato preparation incubated in the presence of a mixture of biosynthetic 14C-labelled terpenyl (mainly farnesyl" cf. Porter et al., ibid., 1963, Io2, 26) pyrophosphates decreased in the order phytoene > phytofluene, ~-carotene > neurosporene > lycopene. [The activity associated with the non-crystalline polyenes was determined by hydrogenation (to perhydrolycopene, or "lycopersane"), chromatography, and subjection of the lycopersane fraction to gas-liquid chromatography- the effluent corresponding to the lycopersane peak being counted: simple chromatography fails to rid the polyenes of radioactive impurities]. (f) Beeler and Porter (Biochem. Biophys. Res. Comm., x962, 8, 367) have demonstrated the conversion of ~4C-labelled phytoene into phytofluene in the tomato. (g) The close structural relationship between the members of the PorterLincoln series, long inferred, has since been made apparent by the elucidation of the precise structures of phytoene, phytofluene, ~-carotene (cf. p. 275 ) and neurosporene (cf. p. 276) (their structures followed from chemical and P.M.R. studies and were confirmed by synthesis: Rabourn and Quawkenbush, and B. C. L. Weedon et al.: cf. Proc. chem. Soc., 1961, 261; most of the structures previously proposed for these compounds were incorrect - cf. Goodwin, Adv. Enzymol., 1959, 2I, 342; the central double bond in both natural phytoene and in natural phytofluene is probably cis: Porter et al., Arch. Biochem. Biophys., 1954, 48, 267; 1965, zxo, 291; Zechmeister, Fortschr. Chem. org. Naturstoffe, 196o, I8, 315): Phytoene

Phytof[uene

~-Carotene

336

THE

CAROTENOID

7

GROUP

r

Thus it now seems certain that the main biosynthetic route to lycopene is, as Porter and Lincoln (p. 334) suggested: phytoene --+ phytofluene --+ ~-carotene > neurosporene ~ lycopene (the "Porter-Lincoln pathway"). (The detection of mono- and bis-dehydrolycopenes in nature (p. 280) suggests that in certain organisms the dehydrogenation process can proceed further" l y c o p e n e - ~ 3,4-dehydrolycopene--~ 3,4: 3',4'-bisdehydrolycopene). Jensen et al. (Biochim. Biophys. Acta, 1958, 29, 477) have demonstrated the existence of another biosynthetic pathway involving dehydrogenation s t e p s - the spirilloxanthin in the bacterium Rhodospirillum rubrum is formed from lycopene through the carotenoid "P48I": lycopene ~ P48I -~ spirilloxanthin. It was also shown that the lycopene originates from phytoene through the Porter-Lincoln pathway, and the additional isolation of colouring matters later (probably) identified (cf. pp. 307 et seq.) as rhodopin, 3,4-dehydrorhodopin, rhodovibrin and OH-spirilloxanthin from the bacterium suggests that the pathway shown in Scheme 22 is followed (Jensen el al., Nature, 1961, z92, 1168; Acta Chem. Scand., 1961, I5, 2058): Phytoene (structure above)

I (Porter-Lincotn pathway )

Neurosporene Lycopene I (.H20,) I

,o

(- 2HI

/

3,4- Oehydrorhodopin

8

BIOSYNTHESIS

OF HYDROCARBONS

337

Me A n h y d r o r h o d 0 v i brin ( . P481 ) ('H203

RhodoVi brin ( - "OH- P4 81" ) (-2H)

~"

M o n o d e m e t h y t a t e d spirilloxanthin ( - "OH-spirit[oxanthin" ? )

M,off ~"

Sp lr it [ oxa nth in

S c h e m e 22.

[The mechanism by which unsaturation is introduced remains uncertain; molecular oxygen is sometimes (cf. p. 333), but not always, required]. The purple bacteria Chromatium strain D and C. vinosum each contain all or most of the above-mentioned compounds and it appears that the same sequence of transformations is occurring in these organisms (C. R. Benedict et al., Biochim. Biophys. Acta, 1961, 54, 525; K. Schmidt, Arch. Mikrobiol., 1963, 46, 127). The spirilloxanthin produced by Rhodopseudomonas gelatinosa cultured in the absence of air probably arises by a rather different pathway which, however, still includes dehydrogenation steps (Scheme 23) (K. E. Eimhjellen and Jensen, Biochim. Biophys. Arta, 1964, 82, 21; K. V. Sunada and R. Y. Stanier, ibid., 1965, lO7, 38) (structures above or on pp. 31o et seq.) (Scheme 23). In aerobic cultures the spheroidene --+ spirilloxanthin sequence is replaced by a parallel sequence involving 2-oxo-derivatives (cf. formula (iii), p. 344), spheroidenone, OH-spheriodenone, 2-oxorhodovibrin, and pigment P518.

338

THE CAROTENOID

Phytoene

(Porter-

Lincoln pathway)

GROUP

7

~__ Neurosporene

I (OH2OO) ChLoroxanthin ( - 2H, methylated ] Spheroidene (

'

Anhydrorhodovibr in

~

(,HEO- ) OH- spheroidene

~

)

(-2H) Rhodovibrin

~ (-2H] Monodeme~hyf.ated $piriUoxanthin

Spirit[ oxanthin

Scheme 23 .

(ii) The cyclisation step Although m a n y of the carotenoids in nature contain cyclic end-groups, it is still not certain at which stage in their biosynthesis cyclisation occurs. Porter and Lincoln (loc. cir.) originally suggested that the following sequence was operating in their specially bred tomatoes (p. 333): Lycopene > 7-carotene > fl-carotene--+ a-carotene

Goodtvin, G. Mackinney and others (Biochem. J., 1953, 53, 531 ; 1961, 80, 48P; Ann. Rev. Plant Physiol., 1961, x2, 230; Arch. Biochem. Biophys., 1954, 53, 479; cf. also H. Yokoyama et al., J. biol. Chem., 1962, 237, 681) later obtained results which suggested that the fl-carotene ill several other systems (including normal, as opposed to hybrid, tomatoes) does not originate from lycopene and 7-carotene but is formed independently of these [possibly by sequential desaturation of cyclic analogues of phytoene, phytofluene, etc. (thereby implying that cyclisation occurs early in the bio-synthetic pathway - this is now thought unlikely)]. Porter's 14C-incorporation experiments suggest that the fl-carotene in tomatoes can be formed through either of two pathways (Scheme 24), the extent to which each route is used varying from one strain to another (Beeler and Porter, 1963, loc. cit.). Various entirely speculative schemes of carotene biosynthesis involving most or all of those carotenes of known structure have been put forward (W. J. Rabourn, Abs. Papers, Amer. Chem. Soc. I32nd Mtg., Sept. 195 7, p. 88C; Y. Mase, J. Vitaminology, I95 9, 5, I6I; A. Winterstein, Angew.

8

BIOSYNTHESIS

339

OF HYDROCARBONS

Phytoene (PO~'h~wera-y,Li:ee~ np. 33(5)

j

Neurosporene

~ f ~ - . ~

.....

Lycopene

~- Z eacarotene .i-

7- Carotene ~'- Carotene S c h e m e 24.

Chem., 196o, 72, 902; Porter and D. G. Anderson, Arch. Biochem. Biophys., 1962, 97, 520) but as yet there is no experimental evidence to indicate which operates and in which environments. It is unlikely that any one carotene is formed by the same route in all plants in which it occurs: K. Decker and H. Uehleke (Z. physiol. Chem., 1961, 323, 61) have demonstrated the conversion of labelled lycopene into fl-carotene in green leaves and the reverse of this in tomato tissue. The common carotenes may be those which lie at the ends of several pathways. The appearance of only traces of certain carotenes in nature does not preclude their lying on a biosynthetic pathway: they may be particularly susceptible to rapid enzymic conversion into the next member of the series. (ttowever, the observation that phytoene, phytoftuene, and ~-carotene appear to be far more widespread in nature than neurosporene (p. 276) suggests that the biosynthetic routes from phytoene to the various fully unsaturated carotenes are common up to the ~-carotene stage and then divide.) The origin of the C,0 skeleton of phytoene, which has been written as the basic precursor of the carotenes in each of the biosynthetic pathways discussed above, will now be considered.

(iii) The origin of the C4o carbon skeleton It has been shown that the two carbon atoms of acetate are incorporated into fl-carotene and lycopene during their biosynthesis in the same regular

34 ~

THE

CAROTENOID

GROUP

7

manner as in other terpenoids. Thus E. C. Grob and R. Bi~tler (Helv., 1956, 39, 1975) isolated the fl-carotene synthesised by the mould Mucor hiemalis growing on media containing (a) [2-14C]- and (b) [I-14C]-acetate and, after dilution with carrier, degraded (cf. pp. 244, 260) samples of the two batches of fl-carotene with: (a) chromic acid (to give 6 moles of acetic acid, one from ]

each C H 3 - - C - ), (b) H202--OsO 4 (to give retinene and fl-ionylideneacet-

~

0

0

~}- Ionytideneacetaidehyde

Retinene

aldehyde), and (c) alkaline permanganate (to give aa-dimethylsuccinic and aa-dimethylglutaric acids). The aldehydes were cleaved with alkali (R. CMe-CH.CHO .~ R.CO.Me + CHz.CHO ), the acetaldehyde so formed then being oxidised to acetic acid. In each instance the carboxyl groups from the acids were converted into carbon dioxide using the Schmidt reaction (R'CO2H + HN3/H * --~ RNH2 + CO2 + N2) and counted as barium carbonate" the methylamine obtained from the acetic acid samples was also counted: the carbon atoms from the methyl group (M) and from the carboxyl group (C) of acetate had been incorporated into the fl-carotene as follows:

M

M

M~c/M'c

- ' ~ - ~ ~ C ~ M ~ C ~ % M ~ C ~ c ~M~c-~c~M%-c ~

C-M/C.M

~

IM

The full labelling pattern in the rings was determined by W. J. Steele and S. Gurin (J. biol. Chem., 196o, 235, 2778) using/5-carotene synthesised by the alga Euglena gracilis growing on [2J4C]- and on [I-14Cl-acetate. Mild oxidation with chromic acid gave acetic acid (4 moles, from the side-chain methyls) and geronic, ~-dimethylglutaric, and (a little) ~-dimethyladipic acids (from the rings):

[~

--- ~

~

2H +

~CO2H 2H

+

j~CO2H ~.,.~CO#

Further degradation of the geronic and c~c~-dimethylglutaric acids gave a series of acid fragments the activities of whose carboxyl carbon atoms (Schmidt reaction) showed that the biosynthetic /~-carotene had been derived from acetate as follows:

8

BIOSYNTHESIS

341

OF HYDROCA R B O N S

M/C'M~C~%JW~ CI. M / ( ~ "

M

J2

A similar incorporation pattern was found in biosynthetic lycopene (see p. 266) (Goodwin and G. D. Braithwaite, Biochem. J., 196o, 76, I), and this was the same as that found earlier in acetate-derived squalene (see p. 343) (J. W. Cornforth and G. Popjdk, ibid., 1954, 58, 403). In addition [ 2 - 1 4 C ] mevalonic acid (below) too was incorporated (and usually with much greater efficiency) into the carotenoids (fl-carotene or lycopene) synthesised by

C.CH2 9CH2OH HO2C.CH/~2~OH IVlevatonic acid

ATP; - C02

C.CH2.CH20- p.P. ~

C---CH.CH20- p.p.

//

/

CH2

CH3

Isopenteny[

pyrophosphate(.ipp,)

~

Oimethy[a|tyl

~ pyrophosphate IPP(- I-I-~07")O

[pp"

~

c~o-

eP.

C~3

~I ~'H3

F --- CH'CH2"CH2'C---~CH'CH20 --P'P" CH3 Ger~qnyl pyrophosphate (C10)

(-H2P207")

/

Farnesyt

pyroph osphate

(C15)

TPNH

~

c 8 9 -P.e

Geranytgeeanyt pyrophosphate (C20) Squatene (C:K))

?

!

"Lycopersene" (C40)

Scheme 25. O|

O|

I I (ATP = Adenosine t r i p h o s p h a t e ; - - P . P . -- p y r o p h o s p h a t e r e s i d u e , - - P - - O - - P - - O H ; T P N H = t r i p h o s p h o p y r i d i n e nucleotide, r e d u c e d form). OII OII (At each step m a r k e d . , one of the h y d r o g e n a t o m s originally a t t a c h e d to C(4 ) of t h e m e v a l o n i c acid is lost as H e ; it is now k n o w n t h a t the e n z y m e s w h i c h effect each of t h e s e steps stereospecifically expels the 4 S h y d r o g e n on each occasion: cf. Corn/orth et al. and Goodwin et al. in Proc. roy. Soc., 1966, 163B, 465, 492, 515).

342

THE CAROTENOID

GROUP

7

tomato fruit, carrot roots, and various micro-organisms (Braithwaite and Goodwin, ibid., 196o, 76, 5, 194; C. O. Chichester et al., J. biol. Chem., 1962, 237, 681; Arch. Biochem. Biophys., 1962, 96, 265; Porter et al., ibid., 196o, 88, 68; Steele and Gurin, 196o, loc. cit.; Grub, Chimia, I957, Ix, 338). Degradation of the fl-carotene revealed a labelling pattern similar to that found earlier (cf. CornJorth et al., Biochem. J., 1958, 69, 146) in mevalonate-derived squalene. The inference was that, just as the triterpenes are formed from squalene (C30), so the carotenoids are formed from the C40 analogue of squalene, "lycopersene", this itself being formed like squalene (cf. L. D. Wright, Ann. Review Biochem., 1961, 30, 525; Popjdk et al., J. biol. Chem., 1961, 236, 1934; 1962, 237, 56; Proc. roy. Soc., 1966, I63B, 465, 492) from mevalonic acid (Scheme 25). Experimental work has since confirmed that carotenoid biosynthesis parallels squalene biosynthesis up to the farnesyl pyrophosphate stage and that it then probably proceeds through to the Cz0 stage as shown. Thus, Chichester et al. (Arch. Biochem. Biophys., 1962, 96, 265; Nature, 1961, 191, 1299 ) have shown that 14C-labelled biosynthetic samples of isopentenyl pyrophosphate (IPP) and farnesyl pyrophosphate are efficiently incorporated into lycopene in tomato homogenate and into fl-carotene in the fungus Phycomyces blakesleeanus, respectively; in addition, the incorporation of the farnesyl pyrophosphate (C15) into the fl-carotene is stimulated when mevalonic acid, a precursor of isopentenyl pyrophosphate (C5), is added to the system. Further, Anderson and Porter (Arch. Biochem. Biophys., 1962, 97,509) found that the addition of more IPP to a carrot-root preparation containing a mixture of labelled biosynthetic (cf. Perter et al., ibid., 1963, io2, 26) farnesyl and isopentenyl pyrophosphates stimulated the production of phytoene (C40)by the system. The formation of geranylgeranyl pyrophosphate from IPP and farnesyl pyrophosphate has been demonstrated in a yeast preparation (K. Kirschner, 1961: cf. Grub, Kirschner and Lynen, Chimia, 1961, I5, 308) and certain other systems (L. W. Wells et al., Fed. Proc., 1964, 23, 426; K. Bloch et al., J. biol. Chem., 1964, 239, 2507). (Further to the above, recent biochemical work using synthetic 14C- and tritium-labelled mevalonic acids strongly suggests that the actual mechanisms of biosynthesis of farnesyl pyrophosphate from mevalonic acid and of geranylgeranyl pyrophosphate from mevalonic acid are chemically identical: cf. Goodwin and Williams, Proc. roy. Soc., 1966, I63B, 515). However, a careful search for lycopersene in ~veral natural carotenoidsynthesising systems has failed to reveal its presence (Goodwin et al., Biochem. J., 1963, 87, 317, 326; Porter et al., Arch. Biochem. Biophys., 1962, 97, 5o9: 1963, io2, 26). All these systems did however contain phytoene (structure below). Similarly, whereas Anderson and Porter's carrot-root preparation

8

BIOSYNTHESIS

343

OF H Y D R O C A R B O N S

(above) did produce z4C-labelled phytoene, no radioactivity could be detected in those fractions which would have contained biosynthetic lycopersene had it been produced (the compounds synthesised by the system were separated chromatographically, synthetic lycopersene being added as marker). The same negative result was obtained using other phytoene-producing systems (Porter et al., 1962, I963, loa. cir. ; Goodwin et al., z963, loc. cir.). Also, whereas the formation of squalene by the condensation of two Cx5 units (farnesyl pyrophosphate and, probably, nerolidyl pyrophosphate: Popjdk et al., loc. tit.) requires the presence of TPNH (as a reducing agent) (F. Lynen et al., Angew. Chem., I958, 70, 738; Goodman and Popjdk, J. Lipid Res., i96o , x, 286), the formation of phytoene from farnesyl and isopentenyl pyrophosphates by Porter's carrot-root preparation does not apparently require TPNH (but does require TPN) (Porter et al., I962, loc. cit.). It appears likely, therefore, that the key step in the formation of the C40 skeleton does not involve concomitant reduction by TPNH so that phytoene rather than lycopersene is the first C4o compound formed: O--RP, Farnesyt pyrophosphate Nerolidyt pyrophosphate TPNH (reduction)

Squalene

(b)

2 ~

c

H

2

~

Gerany[ger~ny[ pyrophosphate

cis

Phytoene

Phytoftuene

or

RR

344

THE

CAROTENOID

GROUP

7

(b) Biosynthesis of xanthophylls (i) The insertion of oxygen functions (---OH, =0, epoxidic O) Although hydroxy-derivatives of the carotenoid precursors neurosporene ("chloroxanthin", p. 31o) and, probably, also of phytofluene (Zechmeister et al., Experientia, 1948, 4, 474; Arch. Biochem. Biophys., 1953, 47, 16o) have occasionally been detected in nature (cf. also Jensen et al., Biochim. Biophys. Acta, 1958, 29, 477), it seems likely that xanthophylls are usually formed by the direct oxidation of the corresponding carotenes (rather than by sequential desaturation of oxy-derivatives of phytoene etc.) (cf. H. Claes, Z. Naturforsch., 1959, x4b, 4; Decker and Uehleke, Z. physiol. Chem., 1961, 323, 61; Jensen, loc. cit. ; B. H. Davies et al., Biochem. J., 1965, 94, 26P). The following specific information is available:

The extent to which 180 is incorporated into the hydroxyl and epoxide groups of the three major xanthophylls (violaxanthin, p. 300" lutein, p. 295" neoxanthin : p. 330) of the alga Chlorella vulgaris when this is grown (a) under 180-enriched oxygen and (b) in 180-enriched water indicates that at least in this organism the hydroxyl oxygen is derived from molecular oxygen and the epoxide oxygen from water (Chichester et al., Arch. Biochem. Biophys., 1962 , 96, 645) (cf. also p. 294 ). (ll)

!

I

Formate can provide the MeO-carbon in spirilloxanthin (p. 308) (Braithwaite and Goodwin, Nature, 1958, i82, 13o4); this and other methoxycarotenoids (pp. 308 et seq.) probably arise by biological methylation (methionine?) of the corresponding hydroxy compounds (cf. Jensen et al., ibid., 1961, 192, 1168). MeO

--

When Rhodopseudomonas spheroides is cultured in the absence of air and then exposed to oxygen the yellow carotenoid "pigment Y" (or "spheroidene": p. 31o) therein is converted (cf. Stanier et al., J. Cell. Comp. Physiol.,

8

BIOSYNTHESIS

345

OF XANTHOPHYLLS

1957, 49, 51) into a red carotenoid ("pigment R", or "spheroidenone"). The oxygen atom is derived solely from molecular oxygen (lso2-incorporation studies: E. A. Shneour, Biochim. Biophys. Acta, 1962, 65, 51o): M

I

Spheroidenone ('R')

Similarly, on exposure of anaerobic cultures of R. gelatinosa to air, the compound OHoy (or OH~ p. 311) therein is converted into OH-R (= OH-spheroidenone), Y is converted into R (cf. above), and spirilloxanthin (p. 308) is converted into the carotenoid P518 (2,2'-dioxospirilloxanthin : p. 311) (K. E. Eimhjellen and Jensen, ibid., I964, 82, 21).

(ii) Miscellaneous postulated pathways (I) Capsorubin, capsanthin, and kryptocapsin [all of which contain a C 5 ring (pp. 322 et seq.) and are produced by red peppers on reaching maturity] are probably formed from the corresponding cyclohexenyl epoxides (violaxanthin, antheraxanthin, and fl-kryptoxanthin-5',6'-epoxide" pp. 300, 299, 3Ol)- possibly through a pinacolic rearrangement of the following type (cf. also p. 294) (L. Cholnoky et al., Acta Chim. Acad. Sci. Hung., 1958, 16, 227):

OH

"Enze'= enzymatic electron acceptor

(2) The isolation of torulene (LXXII), the Cl0 aldehyde (CXXVIII), and torularhodin (CXXIX) (pp. 280, 313,325) from a single micro-organism by A. Winterstein et al. (Ber., 196o, 93, 2951) suggests that these compounds are formed therein from 7-carotene (p. 272) as follows (cf. also neurosporaxanthin, p. 326):

(LXXII)

CliO (CXXVIII)

C02H (CXXIX)

346

THE CAROTENOID GROUP

7

(For evidence that this pathway is followed elsewhere, see F. Kayser and J. Villoutreix, Compt. rend. Soc. Biol., 196o, 154, 1264; 180 tracer work indicates that only one of the oxygen atoms in torularhodin is derived from atmospheric oxygen: K. L. Simpson et al., Biochem. J., 1964, 92, 508). Other speculative biogenetic schemes proposed by Winterstein include (Angew. Chem., 196o, 72, 9o2) 9

~

---

Attylic oxidn.

~---

I

OH Echinenone (p. 316).

Methyl shift Vitamin A2 (p. 288).

Isorenieratene

(p. 277).

Analogies for the last-named transformation are mentioned by Weexlon et al., (J. chem. Soc., I963, 5637" cf. alsoJensen, Acta Chem. Scand., 1965, 19, IO25).