Clifford W. J. Chang
University of West Florida Pensocola, 32504
Marine Natural Products Pigments
An increasing numher of recent publications and symposia dealing with marine chemistry have drawn chemists' attention to this area of research. Previously, marine chemistry was largely restricted to routine analyses as an aid to oceanographers and marine biologists. Current efforts to explore the oceans have given impetus to the quest of discovering potentially useful medicinal compounds. Whereas the nineteen-fifties and early nineteensixties have seen accelerated programs on medicinally active constituents derived from terrestrial plants, a recent conference in Halifax, N. S., sponsored by the Chemical Institute of Canada, viewed the sea as a new frontier of research (I). This conference was hased on the theme "Drugs from the Sea," as were two conferences in 1968 and 1969 a t the University of Rhode Island that were sponsored by the Marine Technological Society. In November 1971 another symposium on the "Physiologically Active Compounds from Marine Organisms" was conducted under the joint auspices of the Marine Science Institutes of the University of South Florida and the University of Miami, and the Florida Academy of Sciences. These symposia had as their goals the dissemination of current knowledge by summarizing research progress, and the demonstration of future potential utility in the field of marine natural products. Introduction
This paper deals with some previous work on marine natural products and provides a review of marine pigments since much progress in this area of research has occurred in the past ten years. Zoologists classify marine invertehrates into several phyla, including some major ones mentioned in parentheses: Protozoa (flagellates, amoeba, foraminifera, etc.); Porifera (sponges); Coelenterata (jellyfishes, sea anemones, corals); Platyhelminthes and Nemertinea (flatworms and rihhonworms); Echinodermata (sea stars, hrittlestars, sea urchins, sea cucumbers, sea lilies); and Mollusca (snails, slugs, bivalves, squids, octopus, etc.). The brilliant colors of many members in the phylum Echinodermata and the relative ease in collecting some of them in shallow waters are perhaps responsible for the relatively extensive knowledge of the chemistry of pigments from this phylum. So far, only the sea urchins have been examined in some detail. The sea urchins, recognized by their short, stubby, or long, slender spines protruding from their oval tests (shells), contain a number of structurally related pigments. The varied hues of the spines, ranging from nearly black, to red, to greenish white, depending on the species, are exceeded only by the bewildering array of colors exhibited when the various niements undereo chromatographic separation. Sea urchins typically yierd compounds havine structures hased on either the nanhthazarin or iugloneskeletons. Additional hydroxyls (e:g., I-V) are f;equently found in the p-positions. A two-carbon side chain, acetyl or ethyl, also appears only in the p-positions, as for example, in I, 11, and V. Crinoids, which belong to a class of the phylum 102 /Journal
of Chemical Education
Figure 1.
Parent compounds of major marine pigment*.
~chinodermata,generally do not afford naphthquinone pigments. The major pigments isolated are hased on the anthraquinone and naphthopyrone structures. Many marine invertehrates, among them, sea anemones, sea stars, sea urchins, afford polyenol pigments as diesters or carotenoproteins. These pigments which are quite labile are usually found as derivatives of the p-carotene structure (see Fig. 1). The Spinochromes
The sea-urchin pigments, not surprisingly, were given the trivial name spinochromes (2) (pigments from spines) although the earliest isolation, and, in fact, the first structural elucidation involved the pigment which is known in the literature as echinochrome A. Echinochrome A is the red pigment first observed by MacMunn in 1885 from the sea urchin Echinus esculentus Linn (3). Some fifty-five years elapsed hefore Kuhn's elegant structural determination (4) and Wallenfels' synthesis (5)confirmed the structure as V. Early work by Lederer (1938-52) (6),Kuroda (1940-67) and Goodwin (1950) (8) was hindered to a large extent by the unsatisfactory methods of isolating the pigment, by heavy emphasis of comhustion data, and by a general dearth of modern spectroscopic tools. The use of alkali metal carbonates onto which the crude pigments were chromatographed followed by column extrusion did not allow the elution of distinct fractions from the column. Interpretation of the comhustion data was often faulty hecause these high-melting compounds were usually solvated with the crvstallizine solvent. and the hieh oxveen ." to carbon content produced problems in the comhustion process. The combination of these factors conseauentlv resulted in reports of large numbers of purportediy dif&rent compounds. In the mid-sixties several groups led by Scheuer in Hawaii. Thomson in Scotland. and Sutherland in Australia started to unwind the wehof uncertainties prevalent hefore that time. These recent structural determinations used the full complement of modem spectroscopic tools, esneciallv nuclear maenetic resonance (nmr) . . and mass spectroscopic techniques, and resorted to sample comparisons and reisolations from orieinal sources. These investigations reduced the large number of supposedly different
(n,
'.&', \I
R3
a,,
I
a
'I
I-P Compound
RI
I I1 111 IV V VI
RI
Spinochrome A OH COCHJ SpinochromeC OH COCHl Spinochrame D OH OH Spinochrome E OH OH Echinochrome A OH OH Spinochrome B .- -. Figure 2. Some commonly o&urring spinochromes.
RB
R4
H OH H OH Et
OH OH OH OH OH
compounds to those shown in Figure 2. The structure of compound VI, a derivative of juglone, was ascertained by Gough and Sutherland (9) in 1964. In the same year Scheuer and co-workers (10, 11) published their results on the compounds I and 11. Based on correlations of comparison samples and reisolations from the spines of sea urchin species obtained from the Mediterranean, California, Australia, Japan, and Hawaii, the Hawaiian group concluded that the commonly occurring spinochro&es found in sea urchins are represented by spinochromes A-E and echinochrome A. independent syntheses of these spinochromes were subsequently reported by Scheuer (12-14) and Thomson (15-17). In 1966 some additional spinochromes were isolated (18) and a semitrivial nomenclature based on the parent jualone and naphthazarin structures became desirable. Moreover, some unusual representatives of echinoderm pigments were uncovered (Fig. 3). 3-Ethyl-2,5,-dihydroxy-
Compound VIII was not the first methoxylated spinochrome isolated from the Echinodermata since Scheuer and co-workers (21) earlier reported the existence of these compounds in the Asteroidea (sea star, Acanthaster planci Linn) and Ophiuroidea (hrittlestars, Ophiocoma ermaceos Muller and Troschel and 0 . insularia Ryman). Moreover, namakochrome XI was reported by Mukai and co-workers (22) to occur in the Holothuroidea (sea cucumber, Polycheria rufescens Brandt) as a protein complex. The chemistry of sea urchin spinochrome pigments appears to he simple since, with the only exception of IX, chirality of these compounds does not enter into the problem of structural elucidation. However, by virtue of the number of possible tautomeric structures in solution, these colorful compounds are interesting and studies by Moore and Scheuer (23) and FariRa and co-workers (24) using nmr data and transacylation reactions have offered a means to oredict the nredominant tautomer in solution. The methodology of spinochrome research is illustrated hv the case historv of soinochrome A. Isolation and separation of spinochromes generally involve dissolution ot the spines with hydrochloric acid, extraction of the free pigments with ether, and column or preparative thicklayer chromatography of the concentrate on acid-washed silica gel, since untreated silica gel is sufficiently basic to react with the acidic spinochromes. The pigment mixture can then he separated by eluting with benzene, chloroform, or mixtures of methanol in chloroform, or ether in benzene. In the elucidation of structures of these pigments the full use of modern spectroscopic tools and transformation products played an important role. For example, mass spectroscopic analysis confirmed the molecular weight of spinochrome A and revealed the presence of an acetyl group. The ultraviolet-visible spectrum indicated a naphthazarin structure. NMR spectroscopy confirmed that the compound dissolved in DMSO-d6 had a methyl singlet of an acetyl function and an aromatic proton singlet. Methoxy derivatives were prepared using both diazomethane and dimethyl sulfate-potassium carbonate. A number of products were isolated in both cases because of partial methylation. The evidence then available was compatible with either XI1 or I as the structure of spinochrome A.
X Flgure 3
Some unusual sp~nochromes
henzoquinone VII and the pyranonaphthazarin IX were isolated from Echinothrix diadema Linn. The latter compound represents the first sea urchin pigment with a side chain ereater than two carbons attached to the naohthazarin skeleton (19). Only recently Thomson (20) reported the Dresence of two methoxvlated s~inochromes.of which one kxample is represented by VIII, and two d i m k c naphthoquinone pigments, of which one example is represented by X , in Diadema antillorurn Phillipi. The source material was obtained from the Caribbean. These later findings are significant since several questions must now he raised. Are these unusual ~ i e m e n t s characteristic of the genus Diadema only? Are somiof the freauentlv isolated s~inochromes(see. . . for example. . . Fie. - 21 . artifacts whose methoxylated precursors may have been hydrolyzed during acid dissolution of the spines? These questions are difficult to resolve since the spines of the echinoids contain the pigment as calcium or magnesium salts and the free pigments are obtained only upon acidification.
The key experiment (10) involved the reaction of spinochrome A with hydrogen chloride saturated in anhydrous methanol in a sealed tube (Fig. 4). The resulting five compounds could he separated by column or preparative thick-layer chromatography with the key compound XIV establishing the structure of spinochrome A as I. Compound XIV, 2,7-dimethoxynaphthazarin, a t that time was unknown in the chemical literature but its structure was deduced by nmr evidence which and nonshowed singlets at 63.94 (-OCHd 66.42 (H), equivalent, hydrogen-bonded protons a t 612.70 and 613.12. On the nmr time scale the four types of protons demonstrate equivalence showing the rapid tautomerism of XIVa =t X N b in deuteriochloroform. Volume 50, Number 2, February 1973 / 103
Loss of the acetyl moiety ortho to the p-hydroxyl from the naphthazarin compound I has precedence (25) and, in fact, this type of cleavage has later been observed in the loss of butyric acid from rhodocomatulin deviatives ohtained from crinoids (36). Dreiding molecular models clearly showed steric crowding about the Cz-hydroxyl and Ca-acetyl functions. A possible mechanism is shown in Figure 5 . Before the structure of spinochrome A was firmly established by the information gained from the spinochrome A-hydrogen chloride-methanol experiment, the probable hiogenesis of the spinochromes was considered. The hiosynthetic pathway for spinochrome A may a prrorr be postulated by a polyketalization of acetate units XVII and in uiuo oxidation of the parent compound XVIII to XIX which eventually yields spinochrome A. This entailed a head to tail condensation pattern (26, 27),the introduction of two peri-hydroxy groups, oxidation, and conversion to the stable hydrogen-bonded form 1.
06
Figure 4. nol.
Reaction of spinochrome A with hydrogen chloride in metha-
Figure 5 . Possible mechanism far the loss of acetyl function in spinochrome A.
With the spinochrome-A structure thus firmly estahlished the author can reflect back to his earlier predoctoral days when two compounds with postulated dissimilar structures were considered identical based primarily on their published melting points and visible-ultraviolet spectra. These two structures are represented by XX and XXI, then known in the literature as spinochrome A and spinochrome M respectively.
co co
/ / C C XVII
COCH,,
XVlIl
I
-XIX
While higher plants may synthesize naphthoquinones via three separate pathways (the homogentisate, the shikimate, and the polyacetate malonate) (29), the only work to-date on the biogenesis of spinochromes suggests that a two-step process involving the acetate pathway may he operative. Lederer and co-workers (28) found a low incorporation of [2-'4CI-acetate in the hiosynthesis of spinochrome E in Arbacia pust~dosaLeske. Such a de nouo synthesis of spinochromes would explain why pigments of this type have either a two-carbon side chain or none a t all. It appears, moreover, that additional work is required in spinochrome hiogenesis since, for example, the question of the origin of the methoxyl carbon can now be raised in light of Scheuer's isolation work (21) and Thomson's recent findings (20). 104 / Journal of
Chemical Education
Structure XX was originally proposed by Kuhn and Wallenfels (30) for spinochrome A and was supported by Lederer and Glaser (31). On chromic acid oxidation one mole of acetic acid was produced and presumably the position of the aliphatic oxygen must therefore be alpha to the aromatic ring. Goodwin and Srisukh (8) reported analytical data in support of the formula C I ~ H I O O SThe . ruby red pigment appeared violet on calcium carbonate (the adsorbent commonly used a t that time) on which it is strongly absorbed. Structure XX was considered tentative by the European workers. The formulation of XXI for spinochrome M was offered after the preparation of several acetate and methylether derivatives (32) and the isolation of glyoxalic acid on ozonolysis of the compound then believed to be a pentaacetate. The use of 50% aqueous-hydrochloric-acid-inmethanol solution by the dapanese workers (33) coupled with our difficulty to interpret the dapanese results suggested to the author to treat his spinochrome pigment with HCl(g) saturated in anhydrous methanol in a sealed tube. The results obtained (described above) coupled with comparisons of Lederer's spinochrome A, Knroda's spinochrome M , and our spinochrome pigment, tentatively la-
beled "H" (for Hawaii) a t that time, thus led to proof of their common identity (10, 11).
Cheeseman, et al.'sl review (41) in which the authors pointed out
The Crinoidal Pigments
The carotenoproteins, which we may define as those proteins in which carotenoids are present in stoichiometric proportions as In contrast to the echinoids (sea urchins) discussed prosthetic groups, constitute a group of compounds.of which reabove, the crinoids (sea lilies) in the Echinodermata yield markably little is known . . . . There are probably thousands of pigments which are readily released into acetone or ethacarotenoproteins to be found in nature. Many have been denol when immersed in these solvents. These animals yield scribed by zoologists but, wlth few exceptions, have at the mast principally non-spinochrome pigments which are not been subjected to simple expermentation. hound as salts in their calcareous skeletons. Attempts to dissolve the tissues with dilute acid produced only traces Indeed, very little work on carotenoproteins appears in of pigments. the chemical literature. The blue chromoprotein, a-crusSutherland's studies (34-36) on Ptilometra austrnlis tacyanin, from the lobster shell has been reported (42) to Wilton and the Comatulo species showed the pigments to have an equivalent weight of 19,000 g chromoprotein/mole be chieflv derivatives of anthraauinone. For examule. . . astaxanthin. The prosthetic group, astaxanthin XXV, is a chemical studies (34) on rhodoptilometrin, isorhodoptilocommon carotenoid isolated from marine sources. It is one metrin. and ntilometric acid culminated manv vears of reof the main pigments isolated from the copepod, Anomsearch dating back to the work of ~ o s e l e ; Hboard the alocera oanatus Sutcliff, which was collected off PensacoChallenper in her cruise to the East Indies in 1874. Mosela Beach in the Gulf of Mexico. Chang characterized it by ley (37)'observed and described spectroscopically the pigits melting point, its parent peak a t m/e 596 and its fragments, " ~ u r n l e ~entacrinin." "red oentacrinin." and mentation pattern in the mass spectrum and by its ultra"antedonin" 'from'some deep-sea crindids. In 1890 Macviolet spectrum (43). From the Holothuroidea Matsuno Munn (38). who is recognized for his ~ i o n e e rwork on sniand coworkers (44) were able to isolate 0.2 mg of astaxan nochrome pigments, also investigaied the orange-red thin from 300 g testes collected from the sea cucumber, ethauolic extract of Antedon macreonema, a crinoid which Holothurra leucosprlota Rrandt. Astaxanthin, characteris now believed to be P. australis Wilton (39). ized bv the usual methods (mixed mn. uv., color reactions Rhodoptilometrin XXII was found to be S(-)-1,6,8with antimony trichloride reagent), was further confirmed trihydroxy - 3 - (1- hydroxy - propyl) - anthraquinone, while bv the isolation of astacene XXVI which is usuallv an arptilometric acid was shown to have structure XXIII (34). tefact obtained readily by air autoxidation of astaxanthin As in the case of spinochrome pigments the rhodomatulin in the presence of base. pigments share two things in common. Both echinoids The above work (44) was undertaken since it was generand crinoids yield quinones-naphthoquinones and anally known that carotenoids as a class are found chieflv thraquinones respectively-which are rarely found in anifrom the integument of asteroids and in the gonads df mals. Also. the isolation of methoxylated pigments appear echinoids. Weedon and Lederer's groups (45) in 1969, to he unusual as they are not frequently found as animal metabolites (21). Further work by Sutherlaud (40) on the crinoid Comantheria perplexa H. L. Clark has revealed the presence of substituted naphthopyrones of which XXIV is one example. Compound XXIV is the sodium salt of the sulfuric ester of 8-hydroxy-5,6-dimethoxy-2-methyl-4H-naphtho[2,3-b] pyran-4-one. The type of compounds isolated thus far suggests that many of these various quinones and pyrones can be patterned according to the Birch polyketalization hypothesis. The pigments of the Comatula species, for example, are so lichen-like that Sutherland was led to remark "the crinoids which are so plant-like in external form have some synthetic capabilities which are typically manifest in plants rather than in animals" (34).
.,
The Carotenoids
The carotenoids constitute the second major class of pigments of marine animals. As variants of the p-carotene structure they normally are complexed as carotenoproteins and as such are quite labile to heat or organic solvents. The current state of knowledge is summarized in
Figure
6.
PHP Same crinoidal pigment3
XXX
Figure 7. Marine carotenoids, some of which are saponification product% e.g.. XXV. XXIX, or oxidation products, e . g . XXVI. XXX. Volume 50. Number 2. February 1973 / 105
upon reinvestigation of earlier work (46, 47), isolated and identified paracentrone XXVII, fucoxanthinol XXVIII. and fucoxanthin from the gonads and epithelium of some 2000 specimans of the sea urchin Paracentrotus liuidus Lamk. Paracentrone, a novel CJI allenic carotenoid, is suggested to arise from fucoxanthinol probably by way of a retro-aldol fission intermediate (47). Figure 7 shows these and other marine cartenoids. An unusual nor-carotenoid, actinioerythrol XXIX, is an artifact of the red pigment, actinioerythrin. Historically, actinioerythrin, obtained from a species of the ~ o e l e n t e r a ta, the sea anemone, Actina equina Linne, was first crystallized by Lederer (48) in 1934. A year later Heilbron and co-workers (49) independently isolated the same compound and a transformation product, violerythrin XXX. Although partial structural work was carried out, it was not until the early nineteen-sixties that the five-memhered ring moieties in carotenoids were recognized. Karrer's group in studies of the red-pepper pigments investigated the nature of the carotenoid capsoruhin end group and suggested a possible pinacol rearrangement intermediate as a precursor in the hiosynthesis from capsanthin (5&52). The structural determinations of actinioerythin and actinioerythrol were established recently hy L.-Jensen and co-workers (53-54) after extensive chemical work involving about thirty derivatives and after a thoroueh examination of their spectroscopic properties. 4ctinio:rythrin is a mixture of diesters consisting of XXIX and lone chain fatty acids of which capric, undecanoie, and lauric acids were identified by gas chromatography-mass spectrometrv. Interestingly, actinioerythrin or actinioerythrol can be smoothly oxidized by oxygen in mild base under controlled conditions to yield violerythrin. The blue violerythrin pigment is a 2,2'-bisnor-derivative of astacene and like astacene is an artifact. Shortly after L.-Jensen's communication (53) on the actinioerythrin pigments anq the possible hiogenesis of these pigments, Weedon communicated (55) his results on the synthesis of violerythrin. The oxidation of astacene with manganese dioxide afforded violerythrin in -10% yield, presumably, involving the intermediate triketone, and the benzilic acid rearrangement compound. The latter compound upon decarboxylation and oxidation would then result in violerythrin (Fig. 8).
Figure 8.
S y n t h e s i s of violerythrin trom
astacene
The biosynthesis of the actinioerythrins is under investigation (541. It should prove to he interesting if astaxanthin is implicated as a precursor since astaxanthin diesters have been isolated along with actinioerythrin in A . equina. The fundamental work of Karrer (52, 56) on the structure of astacene XXVI and the first recognition of a fivemembered ring structural unit in carotenoids have borne fruit in current carotenoid research. The foregoing has been an attempt by the author to select and point out the fascinating type of pigmentary compcunds found in some marine organisms. This paper is by no means exhaustive and excellent reviews by Scheuer (21) and Thomson (57, 58) on spinochrome chemistry, L.-Jensen (59), and Weeden (60) on carotenoid chemistry, and Sutherland (34) on crinoidal anthraquinone chemistry are available as well as a review by Fox 106 /Journal 01 Chemical Education
and Hopkins (61) on the possible physiological function of spinochromes and carotenoids. The development of all aspects of carotenoid chemistry has been updated in a monograph (62) edited by Isler, Guttman, and Solms. This compendium describes the current knowledge of carotenoids since Karrer and Juckers' original text on the carotenoids (63). The author acknowledges the constant encouragement by Professor Scheuer in whose laboratory the work on spinochromes was carried out and the preview of a chapter from his forthcoming book on the chemistry of marine natural products. Literature Cited I l l Chrm. EngNewr,.lune7, 197~.pp.24-5. I21 L4arer.E.. andGlsrer. R.. C. R. Aeod. Sci., Poris. M7.454(1938). 131 MacMunn. C. A,. quart. J Microrcop. S c i . 25. 4691L8851. I4 Kuhn. R.. and Wallmfds, K.. Cham. Be,.. 72, 1407(1939). I51 Wallenfela. K..and Gsuhe. A . Chem. Ber 76.32511941, 161 Ledere.. E.. Biochrm Biophyr Acta, 9.92 119521. and rerersnces cited therein. 171 Kuroda. C.. and Okabms. M.. Pmr. Japan Acod. 43, 41 11967). and referenms cited therein. (81 Gwdw1n.T. W.,and Srisukh. S..Biochrm. J.. 47,69119501. 181 Gough. J..sndSufherland. M U . . T~tmhrdronLrfl..269119641. (10) C h a w C. W. .I.. Maore, R. E.. and Seheuer. P. J., J Amp?. Chem Soc.. 86. 2959
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. ~.
riot" ,.,>"A"" .... .... ....,, .
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