Symposium on Organic Chemistry of Plant Synthesis* SYNTHESIS OF ISOPRENOID COMPOUNDS IN PLANTS JAMES BONNER California Institute of Technology, Pasadena, California
THE isoprenoid G O ~ ~ O Ucomprise ~ ~ S a group of substances which occur widely in plant and animal material, and which have in common the fact that they can be conceived of as built up of elementary units of the 5-carbon branch chain compound isoprene. The naturally occurring isoprenoid compounds
pounds. The experiments here discussed are one part of a general program to discover the chemical processes by which rubber is formed in the plants and were undertaken as a cooperative project by the U. S. Department of Agriculture, Bureau of Plant Industry, Soils and Agricultural Engineering and the California Institute of Technology. Strange as it may seem, essentially CH, no work has been done on the biosynthesis of rubber >-CH=CH~ in the past despite the continued and increasing imCH* portance of natural rubber production. Natural rubinclude the simple terpenes, 10-carbon compounds in ber research has chiefly centered either on the conditions which isoprene units are condensed by twos either necessary for maximum rubber production by plants into chain or into cyclic compounds; the sesquiterpeues, or on the technology of the extracted rubber. An 15-carbon compounds; the diterpenes, 20-carbon com- understandmg of the biochemistry of rubber formation pounds; the triterpenes, 30-carbon compounds; the is not only of intrinsic interest but is of value, also, from the standpoint of application of this knowledge to the synthesis of natural rubber outside of the living TABLE 1 Classesof Naturally OccurringIsoprenoidOrTe~encCom- plant. Given a knowledge of the rubber precursors, pounds Together w i t h Examples of Some of Their Com- the enzymes, and the conditions of polymerization m o n Oxidation Products which obtain in the plant, and ideas may a t least be Example of entertained as to the-possible production synthetically Empi~ical Ezample of ozidatim Twpene c h s fmmula hydrocarbon product of a natural rubber rather than of a rubber substitute. Although the biosynthesis of rubber in the plant has Isoprene C& Monoterpene CloHls Pinene C,~H,~O not heretofore been attacked experimentally, nevertheSesquiterpene ClsH2, Risabolene CIsHesOH farne- less a number of suggestions may be found in the literasol CwHn Camphorone CnoHlsOH vita- ture as to possible courses which such synthesis might Diterpene take. In the first place, it appears unlikely that isomin A Trite'pene CmHa Squalene CsoH~~OHamyrin prene itself is a precursor of rubber. Isoprene has Tetraterpene CmHu Carotenes CloHsOz xrtnthonever been found in ulant tissues even though it has ..~..nI-"Ly"" Polyterpene (C;Hs). Rubber, gutta. been extensively sought, and it appears u n l i i y therefore that this compound is to be considered as an intercarotinoids, includmg carotenes and xanthophylls, mediate. It seems more probable that some 5-carand the high-molecular weight compounds, rubber and bon branched chain compound other than isoprene, but gutta-percha. This discussion will concern itself only possessing the same carbon skeleton, may be the elewith the synthesis of the isoprenoid compound, rubber. mentary unit which is polymerized in the plant to form It is entirely likely, however, in view of the structural rubber. Our investigations thus far deal only with similarities between rubber and the other isoprenoids attempts to discover the nature of the 5-carbon that mechanisms found to apply to the biosynthesis of branched chain precursor of rubber and to discover if rubber may well apply also with appropriate modifica- possible the mechanism by which this 5-carbon tion to the generd synthesis of other isoprenoid com- branch chain compound is formed. The general procedure used consists in the growing of a rubber plant presented before the Division of Orgsnic Chemistry at the or plant part in synthetic nutrient solution, under con115th meeting of the ~ ~chemical~ societyr in s*,, F i ~ trolled ~~ ~ environmental ~~ conditions, so that rubber forcisco, California, March, 1949. mation a t the expense of rubber precursors present in
-
the plant can be controlled with a considerable degree of exactness. To the nutrient solution is added a compound or compounds regarded as being possible rubber precursors or intermediates in rubber synthesis. If the compound causes an increase in the amount of rubber formed by the plant during the short experimental period the compound is then considered as a possible intermediate in rubber synthesis. Final proof that the suspected compound is in fact an intermediate in rubber formation is based on experiments with isotopically tagged molecules of the intermediate. Our experiments deal only with rubber synthesis by a single plant, the guaynle Parthenium argentatum, a shrub native to the desert regions of Southern Texas and Northern Mexico. This plant, which accumulates rubber in the bark of the stem and roots, synthesizes rubber to an extraordinarily high concentration, and guayule plants may in fact contain 20 per cent or more of their total dry weight in the form of rubber. Rubber synthesis may also take place extraordinarily rapidly in the plant and it has been found that during certain times in the life of a guayule plant as much as 50 to 75 per cent of the total dry weight laid down by the plant may consist of rubber. Although rubber is formed abundantly in the bark of the stem and root of guayule it is essentially absent from the leaves. The leaves, on the other hand, are extremely important for the synthesis of rubber by the guayule bark. Thus, if guayule plants are defoliated rubber formation ceases abruptly. It is possible to demonstrate directly that in the leaves substances are formed which are necessary for the formation of rubber by the tissues of the stem. For this purpose, use was made of the tissue culture technique. Small pieces of guayule stem tissue were cut from the plant, treated in such a way as to make them asceptic, and placed upon an asceptic nutrient containing mineral salts, sugar, and other growth factors needed to make the stem tissue grow. Under these conditions the stem tissues grow vigorously in culture, but during the course of this vigorous growth they fail to make any appreciable amount of rubber. If, now, an extract of leaves of guayule plants is added to the asceptic nutrient medium and stem sections cultured upon this medium it is found that the stem sections rapidly accumulate rubber. This effectis brought about only by extracts of leaves of plants actively engaged in rubber formation. Leaves of plants not actively engaged in rubber formation do not have the effect. It is clear, therefore, that under the conditions of rubber synthesis the leaves of the guayule plant produce something which is necessary for rubber formation in the tissues of the stem. The nature of this compound necessary for rubber formation by the stem has not yet been ascertained, and its identification will apparently he moderately complex. In the meantime, however, a fruitful approach to the study of rubber synthesis has been found to lie in the testing of pure synthetic compounds for ability to support rubber formation in such isolated stem cultures. Parallel to the work with stem cultures, experiments have also been carried on with the
synthesis of rubber by guayule seedling. The use of seedlings rather than of mature plants was originally dictated by the necessity of using small amount of the various and often difficulty obtainable compounds to be tested as rubber precursors. The seedlings after germination were transplanted individually to vials containing 15 ml. of nutrient solution previously found to support good growth of such seedlings. The vials were then placed in specially built boxes, so that the entire root system of the plant was maintained in darkness, and these boxes containing plants in groups of 20 placed in the greenhouse under temperature conditions known to be moderately unfavorable to accumulation of rubber by the plant. To the nutrient solution of the seedlings was then added the various compounds suspected of being rubber precursors. With these seedlings, as with the stem tissue cultures, it was found that addition of extract of leaves of guayule plants actively engaged in rubber formation actually increases the amount of rubber formed and accumulated in the stem tissues. Apparently in these seedlings as in the stem tissue cultures from mature plants the limiting factor in rubber formation is the amount of the rubber precursor formed in the leaves. Among the compounds which have the carbon skeleton of isoprene and which are known to appear in nature are isovaleraldehyde, isovaleric acid, tigaldehyde, tiglic acid, and the amino acid, valine. These substances were all tried for ability to support rubber formation in isolated stem tissues or in guayule seedlings and were all found to he inactive. The Russian worker, Prokofieff and others, have suggested that betamethylcrotonaldehyde may he the rubber precursor and that this substance may he formed from the condensation of acetone and acetone and acetaldehyde. Betamethylcrotonaldehyde has not been found in higher plants, but acetone has been isolated from guayule leaves and acetaldehyde is of common occurrence, as is the dimer of hetamethylcrotonaldehyde, citral. The combination of acetone and acetaldehyde, as well as citral, were therefore tested for the ability to support rubber formation in guayule but were found to be ineffective. It has sometimes been suggested also that the simple terpenes, 10-carbon compounds may be precursors of rubber. This hypothesis has certain elements of improbability inherent in it, but nevertheless, it was found that the leaf terpenes of guayule are not capable of supporting rubber formation either in isolated stem tissues or in the seedling test. Our results indicate that the following reaction is of importance in relation to rubber formation: Acetone
+ acetic acid
-
betsmethylorotonic acid
That this reaction, which has not heretofore been suggested in the literature, is involved in rubber formation is indicated by several lines of evidence. Thus, when acetate is added in a concentration of 100 mg. per liter to the nutrient solution in which stem tissues or seedlings are grown, a considerable increase in formation of rubber is brought about. These increases vary from 25 per cent to over threefold in separate experiments
JOURNAL OF CHEMICAL EDUCATION
630 ADD EXTRACT SYNTHETIC MED
rubber, prepared as the ssveral times recrystallized rubber tetrabromide, contained radioactivity at a ' LEAVE3 higher level than the tissue as a whole. In one experiment. studied in particular detail, 75 per cent of the acetate metabolized by the plant appeared in the form of rubber within the period of three days, the remaining 25 per cent of the radioactive acetate metabolized appearing in the form of the various resins formed in guayule and known also to be isoprenoid compounds. 5TEM CULTURE Further evidence that acetone plays a part in this synGROWS ON JYNTHETIC WELL MEDIUM thesis was also obtained by the use of radioactive acenia=smm.tic Rep.es.n*ation of Basic Experiment on Rubb.. Forma- tone prepared from radioactive acetate. When radiotion by Exci-d Stem Tissue of the Guayvle active acetone was supplied to guayule seedlings, radioactivity was recovered in rubber formed by the plant. under different conditions, the variations undoubtedly A critical test of the proposed scheme of ruhber forreflecting variations in the initial status of the tissue. mation is to discover whether or not hetamethylcroIn contrast to the effects of acetate are those of fluor- tonic acid which is able to replace acetate in supporting acetate, which is known from experiments with micro- rubber formation is actually an intermediate in the organisms to be an inhibitor of acetate metabolism. process. Betamethylcrotonic acid has not heretofore Fluoracetate inhibits the effect,of acetate in increasing been widely found in plants and our efforts to isolate this ruhher accumulation. acid have so far been unsuccessful. The following exAcetone, like acetate, exerts an effect on rubber for- periment indicates, however, that hetamethylcrotonic mation by guayule seedlings. Acetone and acetate do acid is formed during the metabolism of acetate by not appear to be additive in their effects with guayule guayule. Guayule plants were fed with radioactive seedlings, but with guayule stem tissue cultures there is acetate. After a period of three days the plants were very considerahle additive effect between the effects of harvested and an appropriate procedure used for obthe two compounds. One further substance, glycerol, taining the organic fraction which should contain was found to influence ruhber accumulation to a con- the betamethylcrotonic acid, if this substance is pressiderable extent. Glycerol is known, however, to be ent in the plant. To this fraction was then added a rapidly converted to acetate and to acetone by various large amount of inactive betamethylcrotonic acid. microbial systems, and it is entirely possible that the This material was then recrystallized from the extract activity of glycerol in rubber formation in plant tissues and found to contain radioactivity. Such radioactivity is due to some such conversion. can only have been at,tained in the presence of radioIn seeking an explanation of the mechanism by which active betamethylcrotonic acid in the plant extract acetate and acetone might effect rubber formation in and seems to indicate strongly that betamethylcrotonic the guayule plant, the possibility was considered that acid is produced in the guayule as a metabolic product betamethylcrotonic acid might arise as a condensation from acetate. product of these two substances. Betamethylcrotonic The experimental results detailed above suggest acid was therefore tested for ability to support ruhber strongly that acetate is an intermediate in rubber synformation, and found to he as active as the combination thesis and that acet,ate may he metabolized to betaof acetate and acetone with both seedling guayule met,hylcrotonic acid, as a further int,ermediate in the plants and with the isolated stem tissue cultures. Suggested intermediate reaotions between acetate and ruhhrr Betamethylcrotonic acid differs in this respect then formation from its dihydro derivative, isovaleric acid, which is CH,COOH inactive. Consideration was further given to the mechanism by which acetone might arise in the tissue. I t was found that acetoacetic acid, known to he an intermediate in the formation of acetone from acetate by various species of the anaerobic bact.erium, Clostn'dium, is active in replacing acetone in rubber formation in tissue of guayule. It thus appears possihle that acetone arises in guayule by t,heintermediacy of acetoacetic acid as it does in ot,her microbial and animal species. These investigations were then extended to the study of the metaholism of radioactive acetate. Acetate was synthesized containing radioactive carbon 14 in both carbon atoms. When this acetate was supplied to process. What now may he considered as possible guayule seedlings or to mature plants, it was found that further reactions in the polymerization of betamethylwithin three days a considerahle portion of the acetate crotonic acid? Inv~stigationson the mechanisms of fatty acid syngiven had appeared in the rubber fract,ion, and that the
A-
DECEMBER, 1949 TABLE 2 Fate of CH,COOH Fed t o Mature Guayule Plants During Active Rubber Accumulation. Time of Exposure t o Ace. tate Is Three Davs. E x ~ t R-3
Fraction Acetate given Stem root Recovered ssscetste Acetate metabolized Recovered as crystalline rubber tetrabromide Reoovered as rosins
+
Total eounfs %of per mzn. aetzvztg ner B olants taken uo
...
2,000,000 5,370 5,082 288
100 94.6 5.4
211 75
3.9 1.4
yo of
acetate metabolized
... ...
.. .
1W
73 26
thesis in microorganisms and in higher animals have shown that this synthesis proceeds stepwise through the condensation of t,wo acet,ate molecules and subsequent
reduction of the product followed by condensation of this fonr-carbon compound with a further acetate molecule, and so on. The present data suggest a farreaching analogy between fatty acid formation and rnbher formation with betamethylcrotonic acid replacing acetate as the fundamental repeating unit. Betamethylcrotonic acid may be coupled with itself to yield a 10-carbon compound, this coupled with a further betamethylcrotonic acid to yield a 15-carbon compound, etc. At each step in such a process, energy would need to be added to the system both to form the new carbon-carbon linkage, and to reduce the residual oxygen atoms present in the structure. These are matters concerning which we have no evidence as yet. Further elucidation of the manner in which the 5-carbon precursor is condensed to rubber can be obtained only in a long-range extension of the present work.
