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The biogenesis of tropic acid in Datura stramonium 1966

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THE BIOGENESIS OF TROPIC ACID IN' DATURA STRAMONIUM by NEIL WAYNE HAMON B.S.P., University of British Columbia, 1964 A THESIS SUBMITTED IN PARTIAL FULFILMENT OP THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN PHARMACY in the Division of Pharmacognosy of the Faculty of Pharmacy We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA July, 1966 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l , make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree t h a t permission., f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood t h a t copying or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g ain s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department of Pharmacy The U n i v e r s i t y of B r i t i s h Columbia Vancouver 8, Canada Date July,1966 i ABSTRACT There have been two pathways postulated for the biogenesis of tropic acid in Datura stramonium. The first of these, involving the amino acid try- 13 ptophan, was proposed by Goodeve and Ramstad . The second utilizes another 15 amino acid! in this oase phenylalanine, and was put forward by Leete and independently by Underbill and Youngken Jr.*^. Since there have been these two mechanisms postulated for the biogenesis of tropic acid in this plant, one must conclude that either the two amino acids act via a common pathway or that there are in fact two separate mechan- isms for tropic acid biogenesis. The purpose of this investigation was to dis- tinguish between these two possibilities. Tryptophan-3-C14, tryptophan-(2-indolyl)-C14', indoleacetic acid-2-C14, phenylalani ne-3-C^, and uniformly ring labelled phenylalanine-!*^, were fed to separate, one week old, sterile, root tissue cultures of Datura stramonium. The time allowed for the metabolism of the radio-active precursor varied from three to twenty-one days. The tissue was then extracted with ethanol and this extract subjected to two-dimensional paper chromatography. Autoradiography was employed to determine the location of labelled metabolites. These meta- bolites were then identified by direct comparison to authentic samples chrom- atographed in an identical fashion. The results of this investigation show that all of the labelled precur- sors do give rise to labelled tropic acid. The pathway from phenylalanine appears to be the predominate mechanism for tropic acid formation in isolated Datura stramonium root tissue. The pathway from tryptophan, although appar- ently playing a minor role in tropic acid biosynthesis in this tissue, was shown to be a unique and independent system for the biogenesis of this arom- atic acid. It is concluded therefore that there are two separate mechanisms involved in the biogenesis of tropic acid in Datura stramonium root tissue. We accept this abstract as conforming to the required standard i i TABLE OF CONTENTS LITERATURE REVIEW 1 INTRODUCTION TO THE PROBLEM 7 METHODS AND MATERIALS 8 RESULTS 19 DISCUSSION OF THE RESULTS 37 CONCLUSIONS 42 BIBLIOGRAPHY 44 i i i LIST OF TABLES I DATA ON THE SOOT TISSUE CULTURE FEEDING EXPERIMENTS 15 I I DATA ON THE VACUUM INFILTRATION EXPERIMENTS 14 I I I RESULTS OF THE TRYPT0PHAN-3-C14FEEDLNO AND INFILTRATION EXPERIMENTS. 20 IV RESULTS OF THE PBENYLALANINE-3-C 1 4 FEEDING AND INFILTRATION EXPERIMENTS 23 V RESULTS OF THE INDOLEACETIC ACID-2-C 1 4 FEEDING AND INFILTRATION EXPERIMENTS 26 VI RESULTS OF THE UNIFORMLY RING LABELLED PBENY3ALANINE-C 1 4 FEEDING EXPERIMENTS 29 VI I RESULTS OF THE TRYPTOPHAN-(2-IND0LYL)-C 1 4 FEEDING EXPERIMENT 32 V I I I SOLVENT SYSTEMS EMPLOYED 34 IX CONFIRMATION OF THE IDENTIFICATION OF SELECTED COMPOUNDS FROM TABLES THREE TO SEVEN 35 X DISTRIBUTION OF THE C 1 4 LABEL IN THE DEGRADATION PRODUCTS OF TROPIC ACID DERIVED FROM THE TRYPT0PHAN-(2-IND0LYL)-C 1 4 FEEDING 36 i v LIST OF FIGURES 1 THE BIOGENESIS OF TROPIC ACID FROM TRYPTOPHAN 5 2 THE BIOGENESIS OF TROPIC ACID FROM PHENYLALANINE 6 3 TYPICAL AUTORADIOGRAM FROM TRYPTOPHAN-3-C*4 FEEDING 21 4 TYPICAL AUTORADIOGRAM FROM PHENYLALANINE-3-C14 FEEDING 24 5 TYPICAL AUTORADIOGRAM FROM INDOLEACETIC ACID-2-C14 FEEDING 27 6 TYPICAL AUTORADIOGRAM FROM UNIFORMLY RING LABELLED PBENYLALANINE-C14 FEEDING 30 7 TYPICAL AUTORADIOGRAM FROM TRYPTOPHAN- (2-IND0LYL)-C14 FEEDING 33 V ACKNOwLKDGMEHT The author would like to take this opportunity to thank the following groups for their interest in this investigation and for the financial assis- tance which they have extended. 1. The Canadian Foundation for the Advancement of Pharmacy 2. The University of British Columbia Committee on Research >(Graduate Studies) 3. Dean Walter H. Gage for the University of British Columbia Graduate Fellowship Award. The author would also like to acknowledge with thanks, Dr. A.M. Goodeve, for his continued interest, encouragement, and guidance, during the course of this investigation. 1. LITERATURE REVIEW L-Hyoscyamine is the most commonly occurring alkaloid in plants of the family Solanaceae. This alkaloid is an ester consisting of a tropinol and a tropic acid portion. The biogenesis of L-hyoscyamine is thought to occur principally in the roots of Datura stramonium and from here the alkaloid is transported via the transpiration stream, through the xylem to the aerial portions of the plant, especially the leaves, where i t is finally stored. There is however, some evidence that L-hyoscyamine can be synthesized in the leaves, especially the young leaves, although in far smaller quantities than that synthesized by the roots. Elucidation of the pathway involved in the biogenesis of the tropinol portion of L-hyoscyamine began with the work of James* in 1949. James carried out experiments involving the feeding of several possible amino acid precursors to detached leaves of Belladonna. He found that M/100 L-arginine and M/lOO ornithine supplied in addition to 1$ sucrose caused increases in the amount of alkaloid produced per leaf both over the initial values at the time of picking and over the value for leaves supplied with 1$ sucrose only. The increases were small but statistically significant. He further found that neither M/lOO L-proline nor M/lOO ammonium sulfate caused similar increases. Glutamic acid, which is a precursor of both ornithine and proline, 2 was investigated by French and Gibson for its possible role in the bio- genesis of hyoscine and hyoscyamine. Glutamic acid in concentrations of 20 and 60 ppm was fed to root tissue cultures of Datura tatula L. French and Gibson conclude that under the conditions and concentrations used in this investigation, L-glutamic acid produces a statistically significant decrease in alkaloid content, although growth rate was improved. 3 Gibson and Abbot investigated proline as a possible precursor for tropinol in Datura tatula L. As in the case of James they conclude that proline is not indicated as a precursor of L-hyoscyamine, but merely enters the metabolic pool as part of synthesized proteins. When these proteins are degraded by the plant some of the activity may then reside in the alkaloid 4 fraction. In a more recent paper, Gibson and Sullivan found that proline is incorporated into tropinol, but that the degree of incorporation is too small for proline to be an immediate precursor of tropinol. Until rather recently i t was thought that a parellel existed bet- ween the biosynthesis of the pyrrolidine ring of nicotine and the tropinol portion of hyoscyamine. The postulated common intermediate was: Investigators such as Dewey, Byerrum and Ball , and Leete , Leete and Liegliud , have shown that when whole tobacco plants (Nicotinia tabacum \ 14 h) are grown on a mediwa containing ornithine-2-C they produce nicotine labelled in positions two and five of the pyrrolidine ring. In similar experiments using Datura stramonium L, Leete, Marion and 8 14 Spenser nave demonstrated that ornithine-2-C is incorporated into 14 hyoscyamine biosynthetically, the C appearing on the bridgehead car- bons. They did not ascertain whether the incorporation of the label was assymetric, and therefore the labelling may nave occurred at position one, five, or at both positions randomly. 3. 9 Later work by Botherner-By, Schutz, Dawson, Scot , and independently by Leete^, indicates that ornithine-2-C^ stereospecifically labels one, but not both of the bridgehead carbons of hyoscyamine* There is no mixture of labelling in both the one and five positions as there had been in the two and five positions of nicotine. This of course indicated that a symmetrical intermediate such as the one postulated for the biogenesis of nicotine could not be involved in the biogenesis of hyoscyamine. In 1964 Leete^ was able 14 to conclude that ornithine-2-C labels only position one of the tropinol portion of hyoscyamine, and has therefore solved the biochemical stereo- specificity of the incorporation of ornithine into tropinol. i 12 14 14 Kaczhowski, Schutte, and Mothes fed acetate-l-C and acetate-2-C to the excised roots of Datura metel. They found that these two compounds are especially incorporated into positions two, three, and four of the tropinol portion of hyoscyamine. Position three is only labelled i f acetate-14 14 1-C is fed, whereas acetate-2-C is not incorporated into this position* The mechanism for this incorporation of acetate into tropinol is thought to involve the condensation of two acetate units to form acetoacetic acid. This molecule then becomes attached through its carbon atom number two to the inter- mediate formed from ornithine. Following this condensation the carboxyl group of the acetoacetic acid is eliminated, leaving carbons two, three, and four derived from acetoacetic acid. There have been two pathways postulated for the biosynthesis of the tropic acid portion of L-hyoscyamine in Datura stramonium. The first of these 13 mechanisms, that proposed by Goodeve and Ramstad , involves the amino acid tryptophan. Whole Datura stramonium plants grown in hydroponic culture were 14 fed tryptophan-3-C and harvested at various time intervals. The radioactive 14 metabolites of tryptophan-3-C were then identified chromatographically. 14 From the results of this work Goodeve was able to propose the pathway out- lined in figure 1 for the biosynthesis of tropic acid from tryptophan. The elucidation of this scheme included the isolation of a l l of the proposed intermediates involved. The most interesting reaction in this metabolic seq- uence is of course the unique cleavage of the indole nucleus to give a free amino group rather than the more conventional and well studied reaction product, an aminoformyl group. The new amino acid, <£-phenyl-^ -amino propionic acid is closely related to phenylalanine, differing only in the positions of the amino and phenyl groups. 4, The second mechanism proposed for the biogenesis of tropic acid was put 15 14 14 forward by Leete . Phenyls! anine-3-C , sodium formate-C , and formaldehyde- 14 C were added to separate nutrient solutions and fed to two month old Datura 14 stramonium plants. He found that phenylalanine-3-C was incorporated into carbon atom number two of the tropic acid portion of hyoscyamine while the sodium formate and formaldehyde gave most of their activity in the tropinol portion of the molecule. He concluded that phenylalanine is a direct pre- cursor of tropic acid. Underbill and Youngken Jr. 1^ investigated further the role of pheny- lalanine in the biogenesis of tropic acid. They administered dl-phenylalanine-14 14 14 3-C , phenylacetic acid-l-C , sodium acetate-2-C , sodium propionate-2-14 14 14 14 C j sodium formate-C , dl-serine-2-C , and dl-zinc lactate-2-C to Datura stramonium L plants grown in hydroponic culture. Phenylalanine—3-14 14 14 C , phenylacetic acid-l-C , and sodium acetate-2-C were the most efficient 14 precursors of hyoscyamine, followed by sodium propionate-2-C , sodium formate-14 14 14 C , and serine-3-C in that order. Zinc lactate-2-C was found to be a 14 poor precursor for hyoscyamine. Administration of phenylalanine-3-C and 14 phenylacetic acid-l-C produced labelling of only the tropic acid portion 14 14 of the molecule, while sodium acetate-2-C , sodium propionate-2-C , and 14 14 sodium formate-C , and serine-3-C labelled almost exclusively the tropinol portion of the molecule. The pathway postulated by Underbill and Youngken Jr. is outlined in figure 2 . It should be noted however that the proposed intermediates have not been isolated and therefore only represent a logical sequence of events to explain the experimental findingSo 17 Grass and Schutte further substantiated the fact that phenylalanine was a good precursor for tropic acid in Datura metel by finding that phenyl- 14 alanine-3-C would act as an intermediate in the biosynthesis of both tropic and benzoic acids in this species. FIGURE 1. 5. NH 2 CH2-CH-C0GH 0 CHo-C-C00H i 2 tryptophan-3-C ,14 CHO indo lepyruv ic a c i d 1 CH2-C00H indo le -3 -a ldehyde i COOH i n d o l e - 3 - a c e t i c a c i d O C 1 COOH -CH &H„ oC - p h e n y l - p - a m i n o p rop ion ic a c i d a t r o p i c a c i d / COOH HgOH t r o p i c a c i d 6. FIGURE 2. rCH -CH-COOH M 2 phenylalanine—3-C 14 O-CH, phenylpyruvic acid HSCoA 2-phenylmalonic semialdehyde tropic acid INTRODUCTION to the PROBLEM In view of the fact that there have been two mechanisms postulated for the biogenesis of tropic acid in Datura stramonium, and further, since these two schemes involve amino acids which have not been reported as being interconvertible, i t was decided to compare the two biosynthetic schemes under identical conditions. The two possibilities which were considered are? 1. That there are in fact two separate mechanisms involved in the formation of tropic acid in Datura stramonium. 2 . That the amino acids involved are first interconverted or at least form a common intermediate which is in turn converted to tropic acid. The purpose of this investigation was therefore to distinguish between these two possibilities. 8. METHODS and MATERIALS Since the purpose of this investigation was to compare two biosynthetic pathways, i t was necessary to maintain easily reproducible, constant, environ- mental conditions. Because of this need plant tissue culture was chosen as the technique to be used since i t fulfilled these requirements and further offered a system which would be free from bacterial and fungal contamination* The use and evaluation of plant tissue culture as an experimental method 18 can be found in review articles prepared by Butcher and Street and by 19 Carew and Staba . The actual methods employed in plant tissue culture are 20 best dealt with by White in his book The Cultivation of Animal and Plant Cells. Excised root tissue cultures were used in this investigation since the biogenesis of L-hyoscyamine is thought to occur primarily in this region of 20 the plant. White's liquid nutrient medium was chosen to support the growth of the isolated root tissue since i t satisfies the hormone requirements 21 of isolated Datura stramonium roots as shown by Bonner • These hormone re- quirements, as determined by Bonner, are nicotinic acid, thiamine, and pyridoxine. White's liquid nutrient medium consists of three parts. They are: an inorganic salt solution, a hormone supplement, and a carbohydrate solution. Preparation of this medium was carried out as follows: Using the best grades of analytical chemicals the following were weighed out: Ca(l0 5) 2 20 gms. MnSÔ  0.45 gms. KagSÔ  20 gms. ZnSÔ  0.15 gms. KC1 8 gms. H,B0, 0.15 gms. J J NaH2P04 1.65 gms. Kt 0.075 gms. These compounds were then dissolved one at a time in sufficient distilled water to produce eight liters .of solution. If the hydrates of any of the salts were used the proper compensations in weights were made. Thirty-six grams of magnesium sulfate were dissolved separately in two liters of dis- tilled water. The two solutions were then mixed slowly and stored under refrigeration. This stock solution is ten times the concentration required in the complete nutrient solution. 9, A second stock solution was prepared by dissolving three hundred m i l l i - grams of thiamine, f i f t y milligrams of nicotinic acid, ten milligrams of pyridoxine, and three hundred milligrams of glycine i n one hundred m i l l i - l i t e r s of dis t i l l e d water. This solution was also stored under refrigeration and i s one hundred times the concentration used i n the complete nutrient solution. In order to prepare two lite r s of the complete nutrient solution, forty grams of sucrose were dissolved in one l i t e r of dist i l l e d water. Then, ten milligrams of ferric sulfate were dissolved i n one hundred m i l l i - l i t e r s of dist i l l e d water, one-half was discarded, and the remainder was added to the sucrose solution. To these, two hundred m i l l i l i t e r s of the stock salt solution and twenty m i l l i l i t e r s of the hormone stock solution were added and the whole mixture was then made up to two li t e r s with distilled water. This medium was then distributed into culture flasks, each flask containing four hundred m i l l i l i t e r s of nutrient. The flasks consisted of one l i t e r erlynmeyer flasks equipped with cotton plugs. When feeding experiments were being done, i t was at this point that the radio-isotopes were added. The culture flasks with their nutrient solution were autoclaved for thirty min- utes at one hundred and twenty degrees centigrade and fifteen pounds pressure. White's liquid nutrient medium was notably deficient in copper and molybdenum. This was not of much importance i n this investigation since there were no symptoms of the deficiency of either mineral and growth was satisfactory. It i s probable that small amounts of both minerals are present as impurities i n the other compounds used to prepare the stock salt solution. The surface of unbroken capsules of Datura stramonium were sterilized by washing them with thirty per cent hydrogen peroxide solution for three to five minutes. The capsules were then broken open and the seeds removed a- septically. The seeds were then shaken vigorously in a stoppered erlynmeyer flask with a further portion of thirty per cent hydrogen peroxide for a period of nine;fcy seconds. Following this sterilization period the seeds were.placed i n previously autoclaved petri dishes Containing f i l t e r paper moistened with sterile d i s t i l l e d water. The petri dishes with the sterile seeds were then placed under an ultra-violet light in order to catalize the degradation of the remaining hydrogen peroxide. When a l l of the hydrogen peroxide had apparently degraded, the petri dishes were placed i n an incub- ator at twenty-eight degrees centigrade and germination was allowed to con- 10, tinue for a period of seven to ten days. Following this period the largest and most rapidly growing roots were excised and placed in culture flasks containing, White's liquid nutrient medium. The averate length for these freshly excised roots was about fwenty-five millimeters. Each one liter culture flask contained twenty excised roots and four hundred milliliters of White's liquid nutrient medium. These root tissue cultures were then placed in an incubator which maintained a constant temperature of twenty-eight degrees centigrade. Growth was allowed to continue for a period of seven days. On the seventh day the excised roots were aseptically transferred to fresh medium, but this medium also contained the radio-isotope which was being fed. Metabolism and growth were again allowed to continue under the conditions previously outlined. The metabolism time allowed before the cul- tures were sacrificed and extracted varied from feeding to feeding. When- ever this period extended beyond seven days the root tissue was placed in fresh nutrient solution containing more of the radio-isotope at the end of each seven day period. Following the period allowed for metabolism the tissue was removed from the culture flask, blotted dry on filter paper, and placed in a glass mortar. A small amount of sand and ethanol were added and the tissue was ground until a thin paste was formed. This paste was then placed into a soxhlet extraction thimble and extracted in a micro-soxhlet apparatus with approximately twenty milliliters of ethanol for a period of six hours. The ethanolic extract thus formed was then evaporated to dryness by allow- ing i t to stand under a stream of air at room temperature. The dried extract was redissolved in a small quantity of ethanol and spotted on eight Inch squares of Whatmann number one filter paper. This chromatography paper was equipped with corner-punched holes suitable for use 22 on Smith's Universal Apparatus . The spotted sample was then subjected to two-dimensional chromatography. The solvent system employed was iso- propanol, ammonium hydroxide, and water (20:1:2) and n-butanol, acetic acid, and water (l2:3s5). The first solvent system was run for fifteen hours. The paper was then allowed to dry in a current of air at room temperature for thirty minutes and then placed in the second solvent system and run until the edge of the chromatogram was reached (approximately six hours). The chromatogram was then removed from the chromatography chamber and again allowed to dry in a stream of air, this time for twenty-four hours. 11. This prolonged drying period is necessary since i t is important that a l l traces of organic solvents are removed before the chromatogram is used in the preparation of auto-radiograms. The location of radio-activity on the chromatograms was accomplished by preparing auto-radiograms. Auto-radiograms were prepared by placing the chromatogram between two sheets of Kodak No-screen Medical X-Bay Film. The film and chromatograms were then placed in an exposure holder for a suitable time period. Following this time period, one of the films was re- moved and developed. The second film was usually left in contact with the radio-active chromatogram for twice the exposure period of the first film. The advantage of having two exposures for each chromatogram is that the first exposure is available for visualization after only a short waiting period while the visualization of radio-active compounds present in only small amounts can be done on the second film. Thus results axe available quickly and accuracy is not sacrificed. The compounds chosen for feeding purposes were: 14 1. tryptophan-5-C 14 2. indoleacetic acid-2-C 3. tryptophan-(2-indolyl)-C14' 14 4. phenylalanine-3-C 5. phenylalanine-C*4 (uniformly ring labelled). These compounds were administered at either the five or ten microcurie per week level. Two additional solvent systems were employed to aid in the identification of radio-active spots which were eluted from the original chromatogram. Ethanol, ammonium hydroxide, and water (16:1:3) and n-propanol, eucalyptol, formic acid, and water (5*5s2 sufficient to saturate) were used in a two- dimensional system for the identification of neutral and acidic spots. Similarly n-butanol, acetic acid, and water (12:3:5) and n-butanol, IN sodium acetate, and IN hydrochloric acid (7:120:60) were used in the id- entification of basic spots. The relative acidities or basicities of the radio-active spots to be rechromatographed were judged by their position on the original chromatogram. Details on the preparation of any of the above •Acknowledgement with thanks is extended to Miss M, Harrison, X-ray technician at the University of British Columbia Health Services, for technical assistance in developing the X-ray films. solvent systems or their use can be found in Smiths book, Chromatographic 22 and Electrophoretic Techniques. Volume I. Details of a l l of the root tissue culture feeding experiments can be found in table 1. The method employed in the production and main- tenance of cultures, extraction of tissues, and the separation and ident- ification of radio-active metabolites was in all cases identical with the methods previously outlined. The administration of three of the radio-active materials was also accomplished by a vacuum infiltration method. In the first of these ex- periments, one seven day old culture of Datura stramonium root tissue was divided into four portions of three hundred milligrams each. These four portions were then treated in the following manner. Two of the portions were placed into separate beakers, each beaker containing ten microcuries of 14 tryptophan-3-C . The remaining two portions were similarly placed into 14 solutions of phenylalanine-3-C • The four beakers were then placed in a bell-jar. The air in the bell-jar was evacuated until bubbles of gas formed in abundance in the solutions contained within the four beakers. The tissue was left inside the bell-jar under this partial vacuum for a period of fifteen minutes. Thirty minutes after removal from the bell-jar, 14 one beaker of tissue containing tryptophan-3-C and one beaker of tissue 14 containing phenylalanine-3-C were emptied into separate glass mortars. These tissues were then sacrificed, extracted, and chromatographed as previously described. The remaining two portions of tissue were treated in a similar fashion after having been allowed to metabolize the radio-active material for one hundred and eighty minutes following removal from the bell-jar. The second experiment involving the use of the vacuum infiltration procedure was done in exactly the same manner. This time the material 14 infiltrated was five microcuries of indoleacetic acid-2-C . The times allowed for metabolism in this instance were fifteen minutes, thirty minutes, sixty minutes, and one hundred and twenty minutes. Data on the two vacuum infiltration experiments can be found in table 2 . 13. TABLE 1. DATA ON THE ROOT TISSUE CULTURE FEEDING EXPERIMENTS LEVEL ISOTOPE ADMINISTERED METABOLISM LEVEL OF ACTIVITY TIME (days) CHROMATOGRAPHED EXPOSURE OF AUTO- RADIOGRAMS (days) PHENILALANINE- 10 uc 7 1700 cpm 10 17 3-C14 5 uc 3 500 cpm 30 90 5 uc 6 500 cpm 30 90 5 uc 9 500 cpm 30 90 5 uc 12 500 cpm 30 90 TRYPTOPHAN- 3-C 10 uc 7 1500 cpm 10 17 5 uc 3 500 cpm 19 90 5 uc 6 500 cpm 30 90 5 uc 9 500 cpm 30 90 5 uc 12 500 cpm 30 90 5 uc 21 1500 cpm 30 90 INDOLEACETIC 5 uc 3 500 cpm 30 90 14 ACID-2-C * 5 uc 6 500 cpm 30 90 5 uc 9 500 cpm 30 90 5 uc 12 500 cpm 30 90 PHENYLALANINE- 5 uc 3 500 cpm 60 90 C 1 4(ring Label- 5 uc 6 500 apm 60 90 led) TRYPTOPHAN- (2-IND0LYL)-C14 5 uc 21 1000 cpm 30 — Note: The level of activity chromatographed was determined by means of a thin window Geiger Tube in direct contact with the chromatogram. 14. TABLE II. DATA OH THE VACUUM INFILTRATION EXPERIMENTS LEVEL METABOLISM LEVEL OF ACTIVITY EXPOSURE OF AUTO- ISOTOPE ADMINISTERED TIME (minutes) CHROMATOOBAPHED RADIOGRAMS (days) TRYPTOEHAN- 10 uo 30 1300 cpm 7 18 3-C14 10 uc 180 1400 cpm 7 18 PBENYLALANINE- 10 uo 30 1400 cpm 7 18 5-C 1 4 10 uc 180 1500 cpm 7 18 INDOLEACETIC 5 uc 15 6000 cpm 15 37 ACID-2-C14 5 uc 30 3000 cpm 15 37 5 uc 60 4600 cpm 15 37 5 uc 120 3000 cpm 15 37 Note: The level of activity chromatographed was determined by means of a thin window Geiger Tube i n direct contact with the chromatogram. 15. The following materials were used as reference compounds to aid in the identification of spots which appeared on the auto-radiograms: Organic acids: y -amino butyric 2-amino-3-phenyl butanoic amygdalic atrolactic atropic benzoic caffeio chlorogenic cinnamic ferulio glutaric glycolic homogentisic p-hydroxycinnamic 5-hydroxyindoleacetic p-hydroxyphenylpyruvic indole-3-acetio indole-3-BL lactic indole-3-propionic kynurenic malonic oxalic phenylacetic o£ -phenyl- ̂  -amino propionic phenyllactic phloretic potassium fumarate pyruvic quinaldic D-quinic shikimic sinapic sodium lactate succinic tropic vanillic Amino acids: ft -alanine L-alanine L-arginine hydrochloride aspartic acid L-cystine 2,4-dihydroxyphenylalanine L-glutamic acid glycine L-histidine L—histidine hydrochloride L-isoleucine L-leucine L-lysine hydrochloride DIfHiiethionine DL-ornithine hydrochloride phenylalanine L-proline DL-serine DL-threonone tryptophan L-tyrosine DL-valine 16. Miscellaneous compounds; DL—alaninol hydrochloride atropine sulfate benzyaldehyde benzyl alcohol hydratropaldehyde hyoscine hydrochloride hyoscyamine 3-indol aldehyde indole indole acetonitrile indolyl-3-acetamide phenethyl alcohol phenethylamine phenylalamine putrescine chloride tryptamine hydrochloride The above reference compounds were dissolved i n ethanol to produce a solution of ten milligrams per m i l l i l i t e r concentration where possible. Compounds which had solubilities i n ethanol lower that this value were dissolved i n other suitable organic solvents. These reference solutions were then spot- ted on chromatography paper i n the same manner as the plant extracts pre- viously mentioned. The amount used for spotting the chromatograms was f i f - teen microliters. Visualization of the reference compounds on the chromato- grams was accomplished using bromphenol blue reagent for the organic acids, ninhydrin-acetic acid reagent for amino acids and amines, sulphanilic acid reagent and iodine reagent for the miscellaneous compounds. A l l of these 2 2 reagents were prepared according to Smith • Atropic acid andX-phenyl- (3 -amino propionic acid were the only two reference compounds not obtained from chemical manufacturers. These two compounds were synthesized i n the laboratory i n the following manner: Atrolactic acid was converted to atropic acid by distillation under 23 reduced pressure according to the method of McKenzie and Wood . At a pres- sure of ten to fifteen millimeters of mercury, five hundred milligrams of atrolactic acid were dist i l l e d . The vapour dist i l l e d at one hundred and eighty degrees centigrade and quickly condensed to form a white crystalline solid. This solid was dissolved i n warm seventy per cent ethanol and hot water was added to this solution until i t became turbid. Upon cooling cry- stals of atropic acid separated. These had a melting point of one hundred six to one hundred seven degrees centigrade which agrees with the value 23 quoted i n the literature . The yield was approximately two hundred m i l l i - grams. 17. <£ -phenyl-/? -amino propionic acid was synthesized by a method outlined 24 by Mannich and Ganz . Sixty grams of diethylphenylmalonate were hydrolyzed by heating at reflux temperature with two hundred m i l l i l i t e r s of a fifteen per cent aqueous sodium hydroxide solution for two hours. The material was then placed i n an ice bath until i t began to freeze and cautiously neutral- ized with concentrated hydrochloric acid. The mixture was made slightly acidic and extracted three times with ether. The ether was allowed to evap- orate spontaneously, leaving behind crystals of phenylmalonic acid. Twenty- seven grams of phenylmalonic acid were chilled i n an ice bath and made faint- l y alkaline with a twenty-eight per cent solution of ammonium hydroxide. A further twenty-seven grams of phenylmalonic acid were then added and the mix- ture was stirred until i t was homogenous. Thirty m i l l i l i t e r s of a thirty- three per cent formaldehyde solution were next added to the mixture which was then stirred for fifteen minutes at zero degrees centigrade. The mix- ture was allowed to stand at room temperature. Decarboxylation began after thirty minutes and crystals began to separate after eighteen hours. The The reaction continued for seventy-two hours. Following this period the crystals of eC-phenyl-^-amino propionic acid were filtered off and re- crystalized in ninety-five per cent ethanol. The yield was approximately fifteen grams of recrystalized material. The observed melting point was two hundred and six to two hundred and ten degrees centigrade. The melting point range found i n the literature was two hundred and two to two hundred 24 and twelve degrees centigrade • Two methods of degradation were employed on the sample of radio-active tropic acid produced i n the tryptophan-(2-indolyl)-C*4 twenty-one day feeding 14 experiment to determine the position of the C label. The sample of tropic acid was eluted from the chromatogram by soxhlet extraction with ether for six hours. The ether was then removed and two hundred milligrams of cold carrier tropic acid were added. Sufficient absolute alcohol was added to the sample of tropic acid to produce twenty m i l l i l i t e r s of solution. This solution was then divided into two equal portions, and both were evaporated to dryness. Decarboxylation of one of the two samples of tropic acid was 27 carried out according to the method outlined by Walling and Wolfstirn . Ten milligrams of fine copper dust and one m i l l i l i t e r of quinoline were added to the sample of tropic acid. The mixture was then heated to two hundred and fifteen degrees centigrade for a period of one hour. The carbon dioxide 18. which was liberated was trapped ia a tenth molar solution of hyamine 10-X manufactured by Rohm and Haas. The styrene produced by the decarboxylation of tropic acid was recovered by acidifying the contents of the reaction flask with dilute hydrochloric acid and extracting this solution with several por- tions of ether. The combined ether extract was washed several times, first with acidified water and finally with dilute sodium hydroxide. The ether was then evaporated to dryness, leaving behind a light brown residue of styrene. The styrene isolated was then weighed in order to determine the percentage yield of the reaction. The second sample of tropic acid was oxidized to benzoic acid by the 16 method described by Underhill and Youngken Jr. . The tropic acid was re- fluxed with five hundred milligrams of potassium permanganate and one hun- dred milligrams of sodium hydroxide in ten milliliters of water for two hours. The hot solution was filtered, acidified with dilute hydrochloric acid, and extracted with several portions of ether. The ether was removed and the residue was subjected to sublimation which produced pure benzoic acid. The melting point of the benzoic acid produced was one hundred and twenty-two degrees centigrade. This agrees with that given by Underhill and Youngken J r . ^ . The benzoic acid isolated was accurately weighed to determine the percentage yield of the reaction. 14 The results of these degradation studies and the percentage of C label at carbon atoms number one, three, and on the benzoic acid portion of the molecule of tropic acid are shown in table X. Control procedures for this investigation included the chromatography of each radio-active compound to establish its purity, unless such information was already supplied by the manufacturer, as well as the chromatography of each radio-active compound after i t had been autoclaved with a small amount of White's liquid nutrient medium for thirty minutes at one hundred and twenty- one degrees centigrade and fifteen pounds pressure. In all cases the radio- active compounds proved to be radio-chemically pure. Extracts were made of the culture media by acicUfying i t slightly with hydrochloric acid and then extracting with ether. The ether solution was ev- aporated to dryness. The dry residue was dissolved in a small amount of eth- anol and treated in the same manner as the extracts of the root tissue cultures. It was found that while small amounts of materials were secreted into the med- ium, these same materials were contained in the extract of the root tissue in greater abundance.. Further investigation into the matter of excretion into the medium was therefore not made. 19. RESULTS The results of the work done on both the plant tissue culture feeding experiments as well as that done on the vacuum infiltration experiments are summarized in tables III to VII. Any compounds which; are listed under the l i s t of reference compounds but not mentioned in these tables were not identified in any of the tissue extracts. Failure of these compounds to appear in the tissue extracts may be due to the fact that; they are not metabolites of the radio-isotope administered, the concentration of these compounds in the tissue is not sufficient for detection, or the solubility in ethanol under the conditions of extraction is not sufficiently high to allow adequate removal from the tissue. Table VIII is a review of the solvent systems used in this series of experiments. Identification of all of the compounds listed in tables III to VII was made by chromatographing the plant extract and the corresponding reference compound in solvent system number one. Further substantiation of the identity of these compounds was made by eluting the radio-active spot with ethanol in a soxhlet apparatus and re-chromatographing this extract with the reference compound in either solvent system number two or three. The results of this work are shown in table IX. 20. TABLE III. RESULTS OF THE TRYPT0PHAN-3-CU FEEDING AND INFILTRATION EXPERIMENTS METABOLISM TIME AFTER COMPOUND ANTICIPATED LENGTH OF FEEDING (days) INFILTRATION (minutes) 3 6 7 9 12 21 30 180 tryptophan + + + + + + + + tryptamine - + + + . + + - + indole-3-aldehyde - - + + + + - + indole acetonitrile - - - - - - - kynurenine + + + + + + + • + alanine - + + + + + + + kynurenic acid - - + + + + - + quinaldic acid - - + + + - skatole - + d -phenyl- § -amino propionic acid — _ + + + + _ atropic acid - - + + + + + + tropic acid + - - phenylalanine - - - - - - - - tryptophanol - - + + + + + + hyoscyamine - - - - - - - - indole-3-DL lactic acid + + + + + + + + indole-3-acetic acid - - - - + + + + indole-3-propionic acid + + 5-hydroxyindole acetic acid _ mm mm mm indole-3-acetamide - - - - - - - - lactic acid — mm — — mm — — — 21. FIGURE 5. DIAGRAM OF TYPICAL AUTORAJIOGRAM FROM TRYPT0PHAN-3-C14 FEEDING butanol, acetic acid, water (12:3:5) Note: see following page for identification of compounds. 22. FIGURE 5 . (continued) C OMPOUND NUMBER IDENTITY 1 tryptophan 2 tryptamine 3 indole-3-aldehyde 4 kynurenine 5 alanine 6 quinaldic acid 7 kynurenic acid 8 oC -phenyl- Q -amino propionic acid 9 atropic acid 10 tryptophanol 11 indole-3-lactic acid 12 indole-3-acetic acid 13 indole-3-propionic acid 23. TABLE IV. RESULTS OF THE PHENHiAIANINE-3-C14 FEEDING AND INFILTRATION EXPERIMENTS METABOLISM TIME AFTER COMPOUND ANTICIPATED LENGTH OF FEEDING (davs) INFILTRATION (minutes) 3 6 7 9 12 30 180 phenylalanine + + + + + + + phenethylamine - + • + + + - + ft-phenyllactic acid - - - - - - - benzoic acid - + + + + + + tyrosine - - - - - - mm p-hydroxyphenyl- pyruvic acid _ _ _ m. mm — — fumaric acid + + + + + + + tropic acid + + + + + + + atropic acid - - - - - - - phenylacetic acid - - + + + + + mandelic acid - - - - - - succinic acid mm - mm mm - - - ct -phenyl-^ -amino propionic acid — _ — _ — — — tryptophan - - - - - mm hyoscyamine - - - + + + phenethyl alcohol - - - mm - - benzyl alcohol - - - - - - - bensaldehyde - - - - - - - lactic acid — — mm MB — mm — 24. FIGURE 4. DIAGRAM QF TYPICAL AUTORADIOGHAM FROM PHEMIALAELNE-3-C14 FEEDING Note: see following page for identification of compounds FIGURE 4. (continued) COMPOUND NUMBER IDENTITY 1 phenylalanine 2 phenethylamine 3 benzoic acid 4 fumaric acid 5 tropic acid 6 phenylacetic acid 7 hyoscyamine 26. TABLE V. RESULTS OF THE INDOLE ACETIC ACID-2-C14 FEEDING AND INFILTRATION EXPF1RTMRMTS METABOLISM TIME AFTER COMPOUND ANTICIPATED LENGTH OF FEEDING (days) INFILTRATION (minutes) 3 6 9 12 15 30 60 120 indole-3-aeetic acid + + + + + + + + indole-3-aldehyde + + + + + + + + indoleacetonitrile mm - - — — — kynurenine - - - - - - — — alanine - - - - mm _ _ _ kynurenic acid - - - - - _ quinaldic acid - - - mm - - — _ skatole - - - mm — _ mm mm cC-phenyl- §> -amino propionic acid _ mm atropic acid + + + + + + + tropic acid - - + + + + + + hyoscyamine - - - + + + + + 5-hydroxyindole- acetic acid mm mm mm indole-5-acetamide - - - mm - - - FIGURE 5, DIAGRAM OF TYPICAL AUTORADIOGRAM FROM INDOLEACETIC ACID-2-C ̂  FEEDING iso-propanol ammonium hydroxide water (20:1:2) O 2 1 butanol, acetic acid, water (12:5:5) Note: see following page for identification of compounds FIGURE 5.(continued) COMPOUND NUMBER 1 2 3 4 5 6 7 IDENTITY indoleacetic acid indole-3-aldehyde quinaldic acid oC-phenyl-j6 -amino propionic acid atropic acid tropic acid hyoscyamine 29. TABLE VI. RESULTS OF THE UNIFORMLY RING LABELLED PHENYLAJAMINE-C14 FEEDING EXPERIMENTS COMPOUND ANTICIPATED LENGTH OF FEEDING (days) 3 6 phenylalanine + + phenethylamine + + phenyllactic acid - - benzoic acid + + tyrosine mm - p-hydroxyphenylpyruvic acid - - fumaric acid . + + tropic acid + atropic acid - - phenylacetic acid - - mandelic acid - - succinic acid mm - d -phenyl- ft -amino propionic acid mm - tryptophan : - - hyoscyamine - + phenethyl alcohol mm - benzyl alcohol - - benzaldehyde - mm lactic acid — 30. FIGURE 6. DIAGRAM OF TYPICAL AUTORADIOGRAM FROM UNIFORMLY RING LABELLED PHEMYLALAHINE-C14 FEEDING FIGURE 6. (continued) COMPOUND NUMBER IDENTITY 1 phenylalanine 2 phenethylamine 3 benzoic acid 4 fumaric acid 5 tropic acid 6 hyoscyamine TABLE VII. RESULTS OF THE TRYPTOPHAN-C2-IND0LYL)-C 1 4 FEEDING EXPERIMENT COMPOUND ANTICIPATED LENGTH OF FEEDING (davs) 21 tryptophan + tryptamine + indole-3 -aldehyde + i ndo le a c e t o n i t r i l e - a lan ine - skato le - oC-pheny l -p -amino prop ion ic a c i d + a t r o p i c a c i d + t r o p i c a c i d + phenylalanine - tryptophanol - hyoscyamine - indo le -3 -DL l a c t i c a c i d + i n d o l e - 3 - a c e t i c a c i d + i n d o l e - 3 - p r o p i o n i c a c i d - 5-hydroxyindole a c e t i c a c i d - indole -3 -acetamide - l a c t i c a c i d mm FIGURE 7. DIAGRAM OF TYPICAL AUTORADIOGRAM FROM TRYPTOPHM-( 2 -IHD0LYL)-C 1 4 FEEDING FIGURE 7. (continued) COMPOUND NUMBER IDENTITY 1 tryptophan 2 tryptamine 3 indole-3 -aldehyde 4 oC-pneny l -p -amino prop ion ic a c i d 5 a t r o p i c a c i d 6 t r o p i c a c i d 7 i n d o l e-3 - l a c t i c a c i d 8 i n d o l e a c e t i o a c i d 34. TABLE VIII. SOLVENT SYSTEMS EMPLOYED SOLVENT SYSTEM NUMBER VERTICAL SOLVENT HORIZONTAL SOLVENT 1. iso-propanol 200 ammonium hydroxide 10 butanol acetic acid (glacial) 120 30 water 20 water 50 ethanol 16 ammonium hydroxide 1 (28$ water 3 butanol 120 acetic acid 30 (glacial) water 50 n-propanol eucalptol formic acid water butanol IN sodium acetate IN hydrochloric acid 5 5 sufficient to saturate 7 120 60 3 5 . TABLE IX. CONFIRMATION OF THE IDENTIFICATION OF SELECTED COMPOUNDS FROM TABLES THREE TO SEVEN TENTATIVE IDENTIFICATION FROM SOLVENT SYSTEM #1 SOURCE OF THE COMPOUND SOLVENT SYSTEM FOR RE-CHROMATOGRAPH CONFIRMATION <£-phenyl-,0 -amino propionic acid tropic acid hyoscyamine fumaric acid fumaric acid lactic acid lactic acid phenylacetic acid tropic acid atropic acid tryptophan-3-C day feeding 14 seven ,14 indole acetic acid-2-C 120 minute infiltration 14 indole acetic acid-2-C 30 minute infiltration 14 phenylalanine-3-C seven day feeding uniformly ring labelled phenylalanine-C six day feeding tryptophan-3-C day feeding 14 seven 14 phenylalanine-3-C seven day feeding 14 phenyl alanine-3-C twelve day feeding tryptophan-(2-indolyl)- C-"-4 twenty-one day feeding tryptophan-(2-indolyl)-C 1 4 twenty-one day feeding 2 2 3 2 2 2 2 2 2 2 36. TABLE X DISTRIBUTION OF TBE C14-LABEL IN THE DEGRADATION PRODUCTS OF TROPIC ACID DERIVED FROM THE TRYPTOPHAN-(2-INDOLYL) -C 1 4 FEEDING COMPOUND f> YIELD OF THE REACTION COUNTS PER MINUTE COUNTS PER MINUTE CORRECTED TO 100% YIELD carbon dioxide 76.87 52.5 68.25 sjbyrene 76.87 65.0 84.5 benzoic acid 86.4 0.0 0.0 Note: The number of counts per minute was corrected to compensate for background radiation and for the efficiency of the apparatus. The scintillation fluid used was Liquiflour* and the samples were counted using a Nuclear-Chicago Unilux liquid s c i n t i l - lation counter. * Liquiflour i s the trade name for a PP0:P0P0P:toluene mixture man- ufactured by Pilot Chemicals, Inc. 37. DISCUSSION OF THE RESULTS The results shown in tables three to seven indicate that there are no intermediates common to both pathways previously outlined for the biogenesis of tropic acid. This does not however, eliminate the possibility of producing 14 14 radioactive tropic acid from tryptophan-3-C , indoleacetic acid-2-C , or tryptophan-(2-indolyl)-C*4 by participation of only the C*4 -carbon atom in the form of a bicarbonate ion. This could be produced by the degradation of 25 14 the labelled precursor. Leete and Louden have shown that phenylalanine-l§C is incorporated into tropic acid, and that the label is almost entirely on the carboxy carbon of tropic acid. The mechanism proposed by these workers is that of an intra-molecular shift, where carbon atom number one of the phenylalanine moves up the chain and becomes attached to carbon atom number three, thus prod- ucing a 2-phenyl propionic acid structure. The net result of this reaction is 26 the same as that found in red clover by Grisebach and Doerr • The biosynthesis of 7-hydxoxy-4,-methoxy-isoflavone in red clover apparently involves an identical shift of the side chain of phenylalanine to give a 2-phenyl propionic acid struc- ture. There is no evidence to support the participation of a bicarbonate ion in this reaction in red clover, however, i t is interesting to note that this type of shift has been found in a plant other than Datura stramonium even i f the actual mechanisms should prove to be different. The reaction in Datura stramonium can apparently utilize carbon dioxide or bicarbonate ion from sources other than carbon atom number one of phenylalanine. This is shown by 14 the fact that phenlyacetic acid-2-C is an efficient precursor of tropic acid. It is possible therefore that bicarbonate produced from the degradation of tryptophan-3-C*4, indoleacetic acid-2-C*4, (by side chain oxidation), and tryptophan-(2-indolyl)-C*4, (by conversion of N-formy kynurenine to kynuren- ine), could be incorporated into the carboxy carbon of tropic acid even though the bulk of the tropic acid atom would have been derived from phenylalanine. If this supposition were true, then feeding sodium becarbonate-C*4 should give rise to radioactive tropic acid with the activity concentrated largely 25 in the carboxy carbon. Leete and Louden have also used this material in their feeding experiments and they found that the tropic acid produced using 14 sodium bicarbonate-C as precursor was randomly labelled rather than carboxy labelled. Because of this, one is led to the conclusion that since tryptophan-14 14 3-C , as shown by Goodeve , labelled only the carboxy carbon of tropic acid 38. i t must act not via a bicarbonate ion but rather by a unique and independent pathway as illustrated by Goodeve in figure one. 14 The results of the tryptophan-3-C feeding experiment are shown in table number three. All of the compounds found in the pathway shown in figure one are found in this table with the exception of indolepyruvic acid. Indolepyruvic acid, as well as being somewhat unstable, is also a very labile compound met- abolically. Because of these two factors i t is not surprising that i t does not appear in the extract. The sequence of appearance of the intermediates of this pathway generally follows that outlined by Goodeve in figure one. The only compound which appears to be out of sequence is indoleacetic acid. It is probable that indoleacetic acid would remain at quite a low and carefully regulated level in root tissue since i t has hormonal activity and generally in- hibits the growth of root tissue. Quite likely the formation of indoleacetic acid is the rate determining step in the biogenesis of t ropic acid from tryp- 14 tophan. When indoleacetic acid-2-C is fed, tropic acid appears labelled 14 after only nine days compared to twenty-one days when tryptophan-3-C is used as the precursor. The sequence of the appearance of intermediates between indoleacetic acid and tropic acid is quite clearly shown in table five. In- dole-3-aldehyde appears after three days of metabolism, <£ -phenyl-0 -amino pro- pionic acid and atropic acid both appear on the sixth day, and tropic acid finally appears on the ninth day. All of these are in the sequence shown in figure one. The results of the twenty-one day feeding experiment using tryp- tophan- (2-indolyl)-C*4 shown in table seven, indicate that the same radio- active intermediates between tryptophan and tropic acid are formed with this 14 compound as had previously been found with the tryptophan-3-C twenty-one day feeding experiment. Conventional cleavage of the indole nucleus of tryptophan would result in the removal of carbon atom number two of the indole nucleus 14 in the form of formate. Since i t is this carbon which carries the C label in tryptophan-(2-indolyl)-C*4l i t was particularly valuable to feed this material and to see that the label was retained in tropic acid. It would be anticipated that this material would give rise to tropic acid specifically labelled in the hydroxymethyl carbon. 14 Both the phenylalanine-3-C and the uniformly ring labelled phenylalanine- 14 C feeding experiments shown in tables four and six respectively, indicate that tropic acid is synthesized within three days. The amount of isotope ut- 59. ilized for tropic acid production, is estimated at approximately thirty per cent, as judged by the intensity of the spot on the autoradiogram. It is evident that the ring of phenylalanine is incorporated into tropic acid as well as the side chain. The speed with which the root tissue forms labelled tropic acid from the ring labelled phenylalanine would indicate that the C is incorporated without degradation of the phenyl ring. The mechanism for the biogenesis of tropic acid from phenylalanine as shown by Underhill and Youngken Jr. in figure two may well be correct in essence. Certainly the scheme is difficult to prove without the isolation of the actual enzymes involved since most of the inter- mediates would remain bound to the enzyme or would be unstable and difficult to isolate. The only discrepancy in this scheme is the fact that the carbon atom number one of phenylalanine is shown to be given off as carbon dioxide. 25 The results of Leete and Louden indicate that this is not the case since carbon atom number one of phenylalanine is incorporated into the carboxy group of tropic acid. This incorporation was found to be higher than that which would be expected i f this carbon atom were cleaved and allowed to enter the general metabolic pool of one carbon fragments and subsequently recovered. It would seem therefore that the mechanism of this reaction is indeed that of an intra-molecular shift where carbon atom number one of phenylalanine is not lost but rather retained to form the carboxy carbon of tropic acid. It is interesting to notice that phenylacetic acid, shown as an alternate precursor for tropic acid in figure two, is not produced from phenylalanine-3-C14 until the seventh day. This acid therefore cannot be considered to be on the pathway between phenylalanine and tropic acid. The absence of labelled atropic acid in these feeding experiments with phenylalanine offers further circumstantial evidence for the existence of two separate and distinct pathways for the biogenesis of tropic acid in Datura stramonium root tissue. The results of the feeding experiments show that labelled hyoscyamine appears from three to six days after the biogenesis of labelled tropic acid when phenylalanine-3-C14, indoleacetic acid-2-C14, and uniformly ring labelled 14 phenylalanine-C , are used as precursors. It would be expected that labelled hyoscyamine would therefore be produced from tryptophan-3-C3"4 and tryptophan- (2-indolyl)-C1 4 i f the feeding experiments were continued for a period of twenty-four to twenty-seven days. 40. The results of degradation studies done on the tropic acid isolated from the tryptophan-(2-indolyl)-C'1'4 twenty-one day feeding experiment are shown in table X. These results show that tryptophan-(2-indolyl)-C14 gave rise to tropic acid which is not only labelled on carbon atom number three, but also on carbon atom number one, the carboxy carbon. The benzoic acid portion of the molecule had very l i t t l e i f any activity. It was an- ticipated that tryptophan-(2-indolyl)-C^4 would produce tropic acid labelled specifically on carbon atom number three. The fact that carbon atom number one carried forty-four percent of the total C 1 4 found in the sample of tropic acid is not at all surprising. Judging from the intensity of the radio- active spots on the autoradiograms produced, i t can be said that approximately ninety percent of the tryptophan-(2-indolyl)-C14 was degraded via the formyl- kynurenine/kynurenine pathway, This of course gave rise to a free formyl-C'*"4 group. First thoughts on this reaction would lead one to believe that this 14 free formyl-C group should produce uniform labelling of the tropic acid 25 14 molecule, just as Leete and Louden obtained when they fed bicarbonate-C to whole plants. The difference of course lies in the fact that isolated root tissue, which was used in these experiments, does not carry on the 25 process of photosynthesis. The work done by Leete and Louden therefore is not comparable, since in their experiments using whole plants, the bicarb- 14 onate-C would be fixed into sugars by photosynthesis. These sugars, es- pecially the trioses and heptoses, serve as direct precursors for the arom- atic amino acids. This therefore leads to uniform labelling of both phenyl- alanine and tryptophan and ultimately to the production of uniformly labelled 25 tropic acid. This is the result obtained by Leete and Louden . The work done with isolated root tissue in this laboratory did lead to quite a dif- ferent result. Carbon atom number three of the tropic acid is labelled by the scheme outlined by Goodeve in figure one. This accounts for fifty-six percent of the total label found on the tropic acid isolated. Forty-four 14 percent of the C was found on carbon atom number one and this presumably was due to the free formyl-C"L4 group produoed from the tryptophan-(2-indolyl) 14 14 -C . This free formyl-C molecule would participate in the biogenesis of tropic acid from phenylacetic acid and in so doing would specifically label carbon atom number one. It is my suggestion that the tropic acid produced 41 from tryptophan-(2-indolyl)-C is really a mixture of tropic acid-l-C , 14 derived from a phenylacetic acid skeleton with the C label originating 14 14 from formate-C , and of tropic acid-3-C derived from tryptophan via the 14 pathway outlined by Goodeve. The benzoic acid derived from the degrada- tion of tropic acid carried no radioactivity. This is in complete agreement with the hypothesis given above. 14 This work done on the position of the C label in the tropic acid produced from tryptophan-(2-indolyl)-C*4 offers direct proof for the indep- endent existence of two separate pathways for the biogenesis of tropic acid in Datura stramonium. 42. CONCLUSIONS The purpose of this investigation was to determine whether tryptophan and phenylalanine acted as precursors for tropic acid via two distinctly sepai*- ate mechanisms or whether these amino acids formed some common intermediate which in turn was responsible for the formation of tropic acid. Prom the results of the work performed in this investigation, i t seems that there are indeed two separate mechanisms responsible for the biogenesis of tropic acid in Datura stramonium root tissue. Judging by the speed at which tropic acid is produced and by the rel- ative amount of tropic acid produced, one would conclude that the pathway from phenylalanine is the more important pathway of the two for the biogenesis of tropic acid. One should bear in mind however, that the results of this invest- igation are based on what may well be to the plant a highly abnormal situation, that of isolated tissue culture. Caution must certainly be used in applying the results of tissue culture experiments to the whole organism. Although plant tissue culture offers many advantages, especially that of reproducability of experimental conditions, recent investigations show that the individual tissue may not behave in the same way, with the same biochemical and physiol- ogical pathways as the entire organism. The emphasis placed by isolated root tissue on the biogenesis of tropic acid from tryptophan is certainly not great. This low emphasis may well be an artifact produced by the experimental method. The biosynthesis of indole- acetic acid in root tissue does not begin to compare in quantity produced to that of the aerial portions of the plant. This could mean that i f tryptophan were fed to intact plants, i t would be transported to the aerial portions of the plant for conversion to indoleacetic acid which would then travel basipetally in constant supply to the root tissue. Certainly when indoleacetic acid is fed to root tissue i t acts as a far better precursor for tropic acid than the amino acid tryptophan. Further studies on the intact plant may show that tropic acid is one of the major metabolites of indoleacetic acid, and that the biogenesis of tropic acid is the major mechanism for the degradation or de- toxification of this plant hormone. In any event, the biogenesis of tropic acid from tryptophan definitely does occur in isolated root tissue. Two pathways exist for the biogenesis of tropic acid in Datura stramonium root tissue grown under aseptic conditions in liquid nutrient media. These two pathways, one from tryptophan and the other from phenylalanine, have been shown to be separate, independent, and distinct. 44. BIBLIOGRAPHY 1. James, ¥.0., New Phytol. 48: 172-185 (1949). 2. French, D.I., Gibson, M.R., J.A.Ph.A. 46: 151-155 (1957). 3. Gibson, M.R., Abbot, B.R., Lloydia 26: 3 (1963). 4. Sullivan, G., Gibson, M.R., J. Ph. Sci. 53: 1058 (1964). 5. Dewey, H.L., Byerrum, R.H., Ball, C,D., Biochem. Biophys. Acta 18: 141 (1955). 6. Leete, E., Chemistry and Industry 537 (1955). 7. Leete, E., Seigfreid, K.J., J. Am. Chem. Soc. 79:4529 (1957). 8. Leete, E., Marion, L., Spencer, E.D., Can. J. Chem. 32: 1116 (1954). 9. Bothner-By, A.A., Schutz, R.S., Dawson, R.F., Scot, H.L., J. Amer. Chem. Soc. 84: 55-57 (1962). 10. Leete, E., J. Amer. Chem. Soc. 84: 52-54 (1962). 11. Leete, E., Tetrahedron Letters 1619 (1964). 12. Kaczkowski, J., Schutte, H.R., Mothes, K., Biochem. Biophys. Acta 46: 588-594 (1961). 13. Goodeve, A.M., Ramstad, E., Experientia 17: 124 (l96l). 14. Goodeve, A.M., Dissertation Abstracts 21: 175 (i960). 15. Leete, E., J. Amer. Chem. Soc. 82: 612-614 (i960). 16. Underhill, E.W., Youngken Jr., H.W. Jr. J. Pharm. Sci. 51: 121 (1962). 17. Grass, D., Schutte, H.R., Archiv. Pharm. 296: 1-6 (1963). 18. Butcher, D.N., Street, H.E., Botanical Review 30: 513-586 (1964). 19. Carew, D.P., Staba, E.J., Lloydia 28: 1 1-26 (1965). 20. White, P.R., The Cultivation of Animal and Plant Cells 2nd ed. The Ronald Press. New York. (1963). 21. Bonner., J., Amer. J. of Botany 27: 692 (1940). 22. Smith, I., Chromatographic and Electrophoretic Techniques Volume I, 2nd ed. William Heinemann Medical Books Ltd, London (i960). 23. McKenzie, A., Wood, J.K., J. Chem. Soc. 115: part two., 834-835 (1919). 24. Mannich, C, Ganz, E,, Berichte der Deutchen Chemischen Gesellschaft 2: 3486-3504 (1922). 25. Leete, E., Louden, M.L., J. Am. Chem. Soc. 84: 1510-1511 (1962). 26. Grisbach, V.H., Doerr, N., Z. Naturforschg. 15b: 284-286 (i960). 27. Walling, C, Wolfstirn, K.B., J, Am. Chem. Soc. 69: 852-854 (1947).


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