THE CHEMISTRY OF THE RED AND BLUE PIGMENTS OF FLOWERS AND FRUITS. PART I
The coloring matters of natural substances have always interested chemists. In cases like indigo and alizarine where by virtue of their accessibility and durability the plant pigments could be utilized for dyeing purposes, the practical value of the substances became so great that almost as soon as a structural theory of organic chemistry had been developed, their chemical constitutions were determined and the materials synthesized. In many other instances, such as the coloring matters of many flowers and fruits which could never be produced in large enough quantity to serve as useful dyes, and where the presence of accompanying substances seriously interfered with the separation of the essential principle, or in which the complexity of this principle impeded the progress of efforts to determine its structure, a precise knowledge of their chemistry has been acquired only in recent years. This does not mean that the subject has failed to attract attention. As long ago as 1664 Robert Boyle published a report on "Experiments and Considerations Touching Colours" in which he examined the color changes which take place when extracts from flowers are treated with acids and alkalies. Many other investigators have studied the subject since then and it has become evident that in a general way the pigments of yellow flowers constitute one class of substances, distinct in chemical character from those of the red and blue flowers which, together, constitute another. The chemistry of the yellow pigments has been well established for some years but real understanding of the second group is quite recent. It is not within the scope of this account to relate the vicissitudes encountered by the many workers in this field prior to 1913: a complete resum6 of such progress as they made can be found in Perkin and Everest's "Natural Organic Colouring Matters." During the last fifteen years, however, remarkable strides in this field have been made by Willstatter, Karrer, and Robinson. In a remarkable series of investigations camed out a t the Kaiser Wilhelm Institute of Dahlem, Berlin, Richard Willstatter laid the foundations for effective progress in a work so masterly that i t received the Nobel prize in chemistry in 1915. Through the efforts of P. Karrer and his students a t the Techniscke Hockschule a t Ziirich, this fundamental work of Willstatter's has been greatly extended and clarified. Both of these men have attacked the problem from the analytical side, first isolating the essential chromatic principle and then effecting a determination of its structure by breaking it up into smaller recognizable fragments. Robert Robinson, a t the University of Manchester, England, hasmeanwhile
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been developing a synthetic technic whose ultimate objective is the synthesis of snbstances identical with the natural red and blue pigments isolated and identified by Willstatter and Karrer. The work of all these men is of iascinating interest and incalculablevalue. Nevertheless so much has been accomplished and the names and formulas of the substances involved appear so forbidding to the uninitiated that many persons who have not followed the work from the beginning are swamped by its apparent complexity. Furthermore, the original papers are reported in three diierent foreign publications, which may not easily be accessible to all and which, aside from the natural consequence that all save Robinson's work is in German, necessarily include a great deal of highly interesting hut somewhat confusing detail. It is the purpose of the present paper briefly to review the accomplishments of Willstatter, Karrer, and Robinson in such an elementary way that a general conception of the present status of our knowledge of the red and blue natural coloring matters of fruits and flowers may be obtained.
The Anthocyanins The term "anthocyan" comes from two Greek roots signifying, respectively, "flower" and "blue," and was introduced by Marquart in 1835 to designate the blue pigments of flowers. We retain other more common uses of one of these roots in "anthology"-a collection of flowers of literature, i. e., a selection of poems: and in "anthomaniaw-an extravagant fondness for flowers. Gradually the belief developed that the red and blue flower pigments were merely different forms of the same substances and that their different colors were due to variation in the character of the cell sap; this resulted in the extension of the term "anthocyan" to all pigments of this group. When it became apparent that in the plant these pigments were combined with sugars and thus occnrred as glucosides, the ending "in" was attached to the word to emphasize this chemical classification. I t is now apparent that the individual anthocyanins contain similar nuclei and that the wide variation in color is due to slight alterations in the molecule which do not affect the main skeleton. Thus the anthocyanins have come to be regarded as a chemical class in the same way as the proteins, fats, or carbohydrates. The anthocyanins are all glucosides and occur as such in the plant cells. They are easily soluble in water or alcohol but insoluble in other organic solvents such as ether or chloroform. For this reason they cannot be extracted from the flowers by means of volatile solvents and special means of separation from the accompanying water-soluble salts and sugars bad to he devised. The anthocyanins are all amphoteric, however, and form salts either with acids or bases. The salts with acids are particularly well crystallized and all the anthocyanins have been isolated by processes in
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JOURNAL OF CHEMICAL EDUCATION
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which, after extraction of the crude pigment and removal of accompanying impurities, the essential principle has been isolated in the form of its salt with hydrochloric acid or occasionally with picric acid. The stable salt formation is in marked contrast to the behavior of the yellow plant pigments of the flavone and flavonol groups whose salts are easily dissociated in solution. It is even more remarkable in view of the fact that the anthocyanins contain no basic nitrogen atom and that these addition products are oxonium salts formed by addition of acid to a divalent oxygen. Despite the quadrivalent oxygen atom the salts are so stable that in many cases they can be heated a t 10O0 without loss of hydrogen chloride. From the structural viewpoint the two most important chemical reactions of the anthocyanins are those with boiling dilute hydrochloric acid and with fused alkalies. Although some twenty-eight definitely characterized anthocyanins have now been isolated, i t was found in every case that upon boiling for a few moments with 207, ( 6 N ) hydrochloric acid the substance was completely hydrolyzed into two components: one a sugar, either glucose, galactose, or rhamnose; the other a sugar-free pigment, hereinafter called an anthocyanidine. According to whether one or two molecules of sugar were found for each molecule of sugar-free pigment, the original anthocyanins are classified into monoglucosides or diglucosides. From a few anthocyanins there was obtained in addition to the carbohydrate and anthocyanidine, one or two molecules of some organic acid, either malonic, p-hydroxyhenzoic, or p-hydroxycinnamicacid in the cases so far discovered. The behavior of the anthocyanin with fused caustic alkali is the same as that of the anthocyanidines presently to be discussed. The sugar-free pigment thus ohtained by acid hydrolysis of each anthocyanin proved to be the real key to the study of their structure. It was found that each anthocyanidine much resembled its parent in general behavior: each formed crystalline salts with acids, gave characteristic colorations with ferric chloride, etc. Moreover, it developed that although the many different individual anthocyanins gave on hydrolysis various kinds or numbers of carbohydrate molecules, the sugar-free pigment obtained was often the same. In fact, from one group of twenty-one different anthocyanins from a wide variety of flowers and fruits, only three different anthocyanidines were obtained and these three proved to differ one from the other only by a single atom of oxygen. Furthermore, the anthocyanidines obtained from the remaining group of seven anthocyanins proved to he merely methyl or dimethyl ethers of these three fundamental sugar-free pigments. Every anthocyanin, therefore, is a compound of an anthocyanidine with one or two molecules of a simple sugar. In a few cases these are also combined with an organic acid, though this is not an essential characteristic. Upon hydrolysis these various components may again he separated.
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The Anthocyanidiies The Structure of the Three Fundamental Anthocyanidines.-The structure of these three fundamental anthocyanidmes, to which reference has just been made, was deduced by Willstatter from the behavior of their chlorides on fusion with sodium hydroxide. Under the degradative action of such treatment each anthocyanidine breaks up into two simpler products, one of which is a phenol, the other a phenol carboxylic acid. The phenol obtained from each of the three homologous anthocyanidines is the same, phloroglucinol (1,3,5-trihydroxy-benzene)(I). The phenol carboxylic acid obtained from the simplest anthocyanidine is p-hydroxybenzoic acid
Phloroglucinol fi-Hydroxybenzoicacid Protocatechuic acid (1)
(11)
(111)
Gallic acid (IV)
(11), that from the next simplest is 3,4-dihydroxybenzoir acid or protocatechuic acid (111), that from the third is 3,4,5-trihydroxybenzoicacid or gallic add (IV). From these observations and the composition of the anthocyanidine chlorides found by analysis, i t was not a long step to deduce the actual structure of the original materials. The simplest was named pelargonidine (V) because it was first obtained from the anthocyanin found in the scarlet pelargonium: the next homolog, isolated from the pigment of the rose, was the first anthocyanidine to be obtained and received the name cyanidine (VI) : the highest homolog was obtained from the anthocyanin from larkspur, otherwise known as delphinium, and so received the name delphinidine (VII). They are conveniently represented in the form of their chlorides.
HOA~O OH
C~aq~oOs. HC1 Pelargonldme chloride
CI~HLOO.. HC!
Cyan~d~ne chlonde
ClsHlo07. HC1 Dclphinidine chloride
While considered separately it might seem that any of these substances represents a fairly complicated organic molecule, yet inspection of the group shows that the three anthocyanidines are respectively mono-, di-, and tri-hydroxy derivatives of a single simpler structural unit. This unit bears the name of :3,5,7-trihydroxyflavyliumchloride (VIII)
3.5.7-TribvdrodavIium chloride . ~
(VIII)
Flavyliwn chloride (2-phenylbenzopyrylium chloride)
Benropyrylium chloride
(nr)
O
and is itself a substitution product of flavylium chloride (IX). The fundamental parent substance of the entire family is benzopyrylium chloride (X) which has been thoroughly studied as a result of the work of Decker and Fellenburg beginning in 1908. This group of anthocyanidines (V, VI, VII), therefore, forms an homologous series where each individual contains one more hydroxyl in the position 2 phenyl group than did the next preceding. It is plain that upon disruption of the substances during alkali fusion the phloroglucinol obtained in each case comes from the left half of the molecule (which is alike for all) : the substituted phenyl group gives rise to the series of hydroxybenzoic acids whose isolation has already been noted. The Synthesis of the Three Fundamental Anthocyanidines.-It is of interest to note at this point that the constitutions thus assigned to these three anthocyanidines by degradative processes have since been brilliantly confirmed by synthesis. Since such syntheses have been independently carried out by entirely different methods in the laboratories of Willstatter in Germany and Robinson in England, and since both the resultant products are identical with the natural material (Ref. I), there no longer seems room for reasonable doubt as to the validity of the assigned fomulas. It is very important to note that these syntheses refer to the several anthocyanidines and not to the glucosidic anthocyanins themselves. Progress toward their synthesis will be mentioned later. The syntheses of pelargonidine, cyanidine, and delphinidine have been approached through the two general methods suggested by the classical work of Decker and Fellenberg for the preparation of benzopyrylium derivatives. These two methods may be summarized briefly as follows: (1) The addition of aryl Grignard reagents to coumarins.
(2) The condensation of o-bydroxybenzaldehydes with appropriate ketones followed by ring closure.
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The first of these general procedures has been used by Willstatter, the second by Robinson. The first anthocyanidine to be prepared was pelargonidine whose synthesis was reported by Willst%ttera t a meeting of the Prnssian Academy of Sciences on July 30, 1914, only a few days prior to the outbreak of the war. The next was cyanidine, reported to the Baeyer Academy of Sciences and the Chemical Society a t Munich in 1920. A formal report of the details of these experiments did not appear until 1924 (Ref. 2). Meantime Robinson's experiments had been going on and his independent synthesis of pelargonidine reported in 1924 (Ref. 3) was rapidly followed by that of cyanidine and delphinidine in the following year (Ref. 4). In 1926 the preparation of synthetic pelargonidine was achieved again (Ref. 5) in a somewhat different way. Willstitter's synthesis of pelargonidine may be indicated as follows: OCHi
I
CH,
CHjO.CH2.CO OH H Phloroglucinaldehyde
CI
cHEOH\"
CHIOCHICOON~ CH~OCH,.CO.O H (Perkin synthesis)
I
CHsNa
(dialomethane1
