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The biogenesis of tropic acid in Datura stramonium Hamon, Neil Wayne 1966-8-16

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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  In presenting for  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 o f the requirements  an advanced degree a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e  t h a t t h e L i b r a r y s h a l l , make i t f r e e l y study.  a v a i l a b l e f o r r e f e r e n c e and  I f u r t h e r agree t h a t permission., f o r e x t e n s i v e  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 g r a n t e d b y t h e Head o f my Department o r by h i s r e p r e s e n t a t i v e s .  I t i s understood that  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 w i t h o u t my w r i t t e n p e r m i s s i o n .  Department o f  Pharmacy  The U n i v e r s i t y o f B r i t i s h C o l u m b i a Vancouver 8, Canada Date  July,1966  copying  g a i n s h a l l n o t be a l l o w e d  i ABSTRACT There have been two pathways postulated for the biogenesis of tropic acid i n Datura stramonium. The f i r s t of these, involving the amino acid try13 ptophan, was proposed by Goodeve and Ramstad . The second utilizes another 15 amino acid! i n 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 i n this plant, one must conclude that either the two amino acids act via a common pathway or that there are i n fact two separate mechanisms for tropic acid biogenesis. The purpose of this investigation was to distinguish between these two possibilities. Tryptophan-3-C , tryptophan-(2-indolyl)-C ', indoleacetic acid-2-C , 14  14  14  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 metabolites were then identified by direct comparison to authentic samples chromatographed i n an identical fashion. The results of this investigation show that a l l of the labelled precursors do give rise to labelled tropic acid. The pathway from phenylalanine appears to be the predominate mechanism for tropic acid formation i n isolated Datura stramonium root tissue. The pathway from tryptophan, although apparently playing a minor role i n tropic acid biosynthesis i n this tissue, was shown to be a unique and independent system for the biogenesis of this aromatic acid. It i s concluded therefore that there are two separate mechanisms involved in the biogenesis of tropic acid i n Datura stramonium root tissue. We accept this abstract as conforming to the required standard  ii  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  iii  L I S T OF TABLES  I  DATA ON T H E SOOT T I S S U E CULTURE FEEDING EXPERIMENTS  15  II  DATA ON T H E VACUUM INFILTRATION EXPERIMENTS  14  III  RESULTS OF T H E TRYPT0PHAN-3-C FEEDLNO AND 14  INFILTRATION EXPERIMENTS.  IV  20  RESULTS OF T H E PBENYLALANINE-3-C  14  FEEDING AND  INFILTRATION EXPERIMENTS  V  23  RESULTS OF T H E INDOLEACETIC A C I D - 2 - C  1 4  FEEDING  AND INFILTRATION EXPERIMENTS  VI  RESULTS OF T H E UNIFORMLY RING LABELLED PBENY3ALANINE-C  VII  14  FEEDING EXPERIMENTS  RESULTS OF T H E TRYPTOPHAN-(2-IND0LYL)-C  VIII  IX  26  29  14  FEEDING EXPERIMENT  32  SOLVENT SYSTEMS EMPLOYED  34  CONFIRMATION  OF T H E IDENTIFICATION OF SELECTED  COMPOUNDS FROM TABLES THREE TO SEVEN X  DISTRIBUTION OF T H E C  1  4  35  L A B E L I N T H E DEGRADATION  PRODUCTS OF TROPIC A C I D DERIVED FROM T H E TRYPT0PHAN-(2-IND0LYL)-C  14  FEEDING  36  iv 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* FEEDING  4  21  4 TYPICAL AUTORADIOGRAM FROM PHENYLALANINE-3-C  14  FEEDING 5  24  TYPICAL AUTORADIOGRAM FROM INDOLEACETIC ACID-2-C FEEDING  27  14  6 TYPICAL AUTORADIOGRAM FROM UNIFORMLY RING LABELLED PBENYLALANINE-C FEEDING 14  30  7 TYPICAL AUTORADIOGRAM FROM TRYPTOPHAN(2-IND0LYL)-C FEEDING 14  33  V  ACKNOwLKDGMEHT  The author would like to take this opportunity to thank the following groups for their interest i n this investigation and for the financial assistance 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 biogenesis 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 between 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 i s 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 i s 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 stereospecificity 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 i s only labelled i f acetate14 14 1-C i s fed, whereas acetate-2-C i s not incorporated into this position* The mechanism for this incorporation of acetate into tropinol i s thought to involve the condensation of two acetate units to form acetoacetic acid. This molecule then becomes attached through i t s carbon atom number two to the intermediate formed from ornithine. Following this condensation the carboxyl group of the acetoacetic acid i s 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 f i r s t 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 i n this metabolic sequence i s 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 i s 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 formaldehyde14 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 i n the tropinol portion of the molecule.  He concluded that phenylalanine i s a direct pre-  cursor of tropic acid. Underbill and Youngken J r . ^ investigated further the role of pheny1  lalanine i n the biogenesis of tropic acid. They administered dl-phenylalanine14 14 14 3-C , phenylacetic acid-l-C , sodium acetate-2-C , sodium propionate-214 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—314 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 formate14 14 14 C , and serine-3-C i n 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. i s outlined i n 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 i n Datura metel by finding that phenyl14 alanine-3-C  would act as an intermediate i n the biosynthesis of both tropic  and benzoic acids i n this species.  5. FIGURE 1.  NH CH -CH-C0GH  0 CH -C-C00H i 2  2  o  2  tryptophan-3-C  ,14  indolepyruvic acid  1 CH -C00H  CHO  2  indole-3-aldehyde  indole-3-acetic  i  O  acid  COOH -CH C &H„  COOH  1  oC - p h e n y l - p - a m i n o p r o p i o n i c acid  atropic acid  / COOH HgOH  tropic acid  6.  FIGURE 2.  rCH -CH-COOH M 2  phenylalanine—3-C 14  O  -CH,  phenylpyruvic a c i d  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 i n 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 i s 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, environmental 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 i n 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  i n his book The Cultivation of Animal and Plant  Cells. Excised root tissue cultures were used i n this investigation since the biogenesis of L-hyoscyamine i s thought to occur primarily i n 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 requirements, 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  KagSO^ KC1  2  20 gms.  MnSO^  0.45 gms.  20 gms.  ZnSO^  0.15 gms.  8 gms.  H,B0,  0.15 gms.  J  NaH P0 2  4  1.65 gms.  Kt  J  0.075 gms.  These compounds were then dissolved one at a time i n sufficient distilled water to produce eight liters .of solution. I f the hydrates of any of the salts were used the proper compensations i n weights were made. Thirty-six grams of magnesium sulfate were dissolved separately i n two liters of distilled water. The two solutions were then mixed slowly and stored under refrigeration.  This stock solution i s 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 d i s 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 l i t e r s of the complete nutrient solution, forty grams of sucrose were dissolved i n one l i t e r of d i s t i l l e d water. Then, ten milligrams of f e r r i c sulfate were dissolved i n one hundred m i l l i l i t e r s of d i s t 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 s a l t  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 l i t e r s with d i s t i l l e d 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 f o r thirty minutes at one hundred and twenty degrees centigrade and f i f t e e n pounds pressure. White's l i q u i d nutrient medium was notably deficient i n 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.  I t 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 s t e r i l i z e d by washing them with thirty per cent hydrogen peroxide solution f o r three to five minutes. septically.  The capsules were then broken open and the seeds removed a-  The seeds were then shaken vigorously i n a stoppered  erlynmeyer  flask with a further portion of thirty per cent hydrogen peroxide for a period of nine;fcy seconds.  Following this s t e r i l i z a t i o n period the seeds  were.placed i n previously autoclaved petri dishes Containing f i l t e r paper moistened with s t e r i l e d i s t i l l e d water.  The petri dishes with the s t e r i l e  seeds were then placed under an ultra-violet l i g h t i n 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 incubator 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 l i t e r 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 cultures were sacrificed and extracted varied from feeding to feeding. Whenever 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 f i l t e r 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 i n 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 allowing i t to stand under a stream of air at room temperature. The dried extract was redissolved i n a small quantity of ethanol and spotted on eight Inch squares of Whatmann number one f i l t e r 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 isopropanol, ammonium hydroxide, and water (20:1:2) and n-butanol, acetic acid, and water (l2:3s5). The f i r s t solvent system was run for fifteen hours. The paper was then allowed to dry i n a current of air at room temperature for thirty minutes and then placed i n 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 i n a stream of air, this time for twenty-four hours.  11. This prolonged drying period i s necessary since i t i s important that a l l traces of organic solvents are removed before the chromatogram i s used i n 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 i n an exposure holder for a suitable time period. Following this time period, one of the films was removed and developed.  The second film was usually left i n contact with the  radio-active chromatogram for twice the exposure period of the f i r s t film. The advantage of having two exposures for each chromatogram i s that the first exposure i s available for visualization after only a short waiting period while the visualization of radio-active compounds present i n only small amounts can be done on the second film.  Thus results axe available  quickly and accuracy i s not sacrificed. The compounds chosen for feeding purposes were: 14 1. tryptophan-5-C 14 2. indoleacetic acid-2-C 3. tryptophan-(2-indolyl)-C ' 14 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 i n 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 i n a twodimensional 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 i n the i d 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 i s 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 i n table 1. The method employed i n the production and maintenance of cultures, extraction of tissues, and the separation and identification of radio-active metabolites was i n a l l 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 f i r s t of these experiments, 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 i n 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 bell-jar.  • The four beakers were then placed i n a  The air i n the bell-jar was evacuated until bubbles of gas  formed i n abundance i n 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 i n  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 i n exactly the same manner. This time the material 14 infiltrated was five microcuries of indoleacetic acid-2-C  . The times  allowed for metabolism i n 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 i n table 2 .  13. TABLE 1. DATA ON THE ROOT TISSUE CULTURE FEEDING EXPERIMENTS  LEVEL ISOTOPE  METABOLISM LEVEL OF ACTIVITY  ADMINISTERED TIME (days) CHROMATOGRAPHED  PHENILALANINE-  EXPOSURE OF AUTORADIOGRAMS (days)  10 uc  7  1700 cpm  10  17  5 uc  500 cpm  30  90  5 uc  3 6  500 cpm  30  90  5 uc  9  500 cpm  30  90  5 uc  12  500 cpm  30  90  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  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  PHENYLALANINE-  5 uc  3  500 cpm  60  90  C (ring Labelled)  5 uc  6  500 apm  60  90  TRYPTOPHAN-  5 uc  21  1000 cpm  30  —  3-C  14  TRYPTOPHAN3-C  INDOLEACETIC 14 ACID-2-C *  14  (2-IND0LYL)-C  14  Note: The level of activity chromatographed was determined by means of a thin window Geiger Tube in direct contact with the chromatogram.  14. TABLE I I . DATA OH THE VACUUM INFILTRATION EXPERIMENTS  LEVEL ISOTOPE  ADMINISTERED  TRYPTOEHAN-  METABOLISM  LEVEL OF ACTIVITY  TIME (minutes) CHROMATOOBAPHED  EXPOSURE OF AUTORADIOGRAMS (days)  10 uo  30  1300  cpm  7  18  10 uc  180  1400  cpm  7  18  10 uo  30  1400  cpm  7  18  10 uc  180  1500  cpm  7  18  INDOLEACETIC  5 uc  15  6000 cpm  15  37  ACID-2-C  5 uc  30  3000 cpm  15  37  5 uc  60  4600 cpm  15  37  5 uc  120  3000 cpm  15  37  3-C  14  PBENYLALANINE5-C  Note:  14  14  The level of a c t i v i t y 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 i n the identification of spots which appeared on the auto-radiograms: Organic acids: y -amino butyric  kynurenic  2-amino-3-phenyl butanoic  malonic  amygdalic  oxalic  atrolactic  phenylacetic  atropic  o£ -phenyl- ^ -amino propionic  benzoic  phenyllactic  caffeio  phloretic  chlorogenic  potassium fumarate  cinnamic  pyruvic  ferulio  quinaldic  glutaric  D-quinic  glycolic  shikimic  homogentisic  sinapic  p-hydroxycinnamic  sodium lactate  5-hydroxyindoleacetic  succinic  p-hydroxyphenylpyruvic  tropic  indole-3-acetio  vanillic  indole-3-BL lactic indole-3-propionic Amino acids: ft -alanine  L-leucine  L-alanine  L-lysine hydrochloride  L-arginine hydrochloride  DIfHiiethionine  aspartic acid  DL-ornithine hydrochloride  L-cystine  phenylalanine  2,4-dihydroxyphenylalanine  L-proline  L-glutamic acid  DL-serine  glycine  DL-threonone  L-histidine  tryptophan  L—histidine hydrochloride  L-tyrosine  L-isoleucine  DL-valine  16.  Miscellaneous compounds; DL—alaninol hydrochloride  indole  atropine sulfate  indole acetonitrile  benzyaldehyde  indolyl-3-acetamide  benzyl alcohol  phenethyl alcohol  hydratropaldehyde  phenethylamine  hyoscine hydrochloride  phenylalamine  hyoscyamine  putrescine chloride  3-indol aldehyde  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 s o l u b i l i t i e s i n ethanol lower that this value were dissolved i n other suitable organic solvents. These reference solutions were then spotted on chromatography paper i n the same manner as the plant extracts previously mentioned. The amount used f o r spotting the chromatograms was f i f teen microliters.  Visualization of the reference compounds on the chromato-  grams was accomplished using bromphenol blue reagent f o r 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 22  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 d i s t i l l a t i o n under 23 reduced pressure according to the method of McKenzie and Wood  . At a pres-  sure of ten to f i f t e e n millimeters of mercury, five hundred milligrams of atrolactic acid were d i s t i l l e d .  The vapour d i s t i l l e d at one hundred and  eighty degrees centigrade and quickly condensed to form a white crystalline solid.  This s o l i d was dissolved i n warm seventy per cent ethanol and hot  water was added to this solution u n t i l i t became turbid. stals of atropic acid separated.  Upon cooling cry-  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 grams.  . The y i e l d was approximately two hundred m i l l i -  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 f o r two hours.  The material was  then placed i n an i c e bath u n t i l i t began to freeze and cautiously neutralized with concentrated hydrochloric acid.  The mixture was made s l i g h t l y  acidic and extracted three times with ether. The ether was allowed to evaporate spontaneously, leaving behind crystals of phenylmalonic acid.  Twenty-  seven grams of phenylmalonic acid were c h i l l e d i n an i c e bath and made f a i n t 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 mixture was stirred u n t i l i t was homogenous. Thirty m i l l i l i t e r s of a t h i r t y three per cent formaldehyde solution were next added to the mixture which was then stirred for f i f t e e n minutes at zero degrees centigrade. ture was allowed to stand at room temperature.  The mix-  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 f i l t e r e d o f f and recrystalized i n ninety-five per cent ethanol. The y i e l d was approximately f i f t e e n grams of recrystalized material.  The observed melting point was  two hundred and s i x 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* twenty-one day feeding 4  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 f o r 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 i a 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 portions of ether. The combined ether extract was washed several times, f i r s t 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 i n 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 refluxed with five hundred milligrams of potassium permanganate and one hundred 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 Jr.^.  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 i t s 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 twentyone degrees centigrade and fifteen pounds pressure. In a l l cases the radioactive 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 evaporated 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 medium, these same materials were contained in the extract of the root tissue i n 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 i n tables III to VII. Any compounds which; are listed under the l i s t of reference compounds but not mentioned i n these tables were not identified i n 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 i n the tissue i s not sufficient for detection, or the solubility i n ethanol under the conditions of extraction i s not sufficiently high to allow adequate removal from the tissue. Table VIII i s a review of the solvent systems used i n this series of experiments. Identification of a l l of the compounds listed i n tables III to VII was made by chromatographing the plant extract and the corresponding reference compound i n 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 i n 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-C FEEDING AND INFILTRATION EXPERIMENTS U  METABOLISM TIME AFTER COMPOUND ANTICIPATED  LENGTH OF FEEDING (days) 3 6 7 9 12 21  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  +  -  + + +  +  +  +  +  -  + + _  +  +  +  +  +  +  +  +  -  -  -  -  +  +  indole-3-acetamide  - - - - -  -  -  mm  lactic acid  —  —  —  —  mm  mm  INFILTRATION (minutes) 180 30  —  —  mm  -  •  21. FIGURE 5. DIAGRAM OF TYPICAL AUTORAJIOGRAM FROM TRYPT0PHAN-3-C FEEDING 14  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 6  alanine  7  kynurenic acid  8  quinaldic acid 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-C  14  FEEDING AND INFILTRATION EXPERIMENTS  METABOLISM TIME AFTER COMPOUND ANTICIPATED  LENGTH OF FEEDING (davs) 6 12 7 3 9  phenylalanine  +  +  +  +  +  INFILTRATION (minutes) 180 30  +  +  tyrosine  - +• + + - - - - + + + - - - -  p-hydroxyphenylpyruvic acid  _  _  _  m.  mm  —  —  fumaric acid  +  +  +  +  +  +  +  tropic acid  +  +  +  +  +  +  mm  mm  -  + -  +  —  _  - - + - - -  —  —  -  -  —  mm  —  mm  phenethylamine ft-phenyllactic  acid  benzoic acid  atropic acid phenylacetic acid mandelic acid succinic acid ct -phenyl-^ -amino propionic acid tryptophan hyoscyamine phenethyl alcohol benzyl alcohol bensaldehyde l a c t i c acid  - - - - - + + - - mm  —  _  -  -  —  —  mm  MB  +  + + +  -  -  +  -  +  -  + mm  + mm  +  —  24.  FIGURE 4. DIAGRAM QF TYPICAL AUTORADIOGHAM FROM PHEMIALAELNE-3-C FEEDING 14  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-C  14  FEEDING AND INFILTRATION EXPF1RTMRMTS  METABOLISM TIME AFTER COMPOUND ANTICIPATED  LENGTH OF FEEDING (days) 6 12 3 9  indole-3-aeetic acid  +  indole-3-aldehyde  +  indoleacetonitrile  mm  kynurenine alanine kynurenic acid quinaldic acid skatole cC-phenyl- §> -amino propionic acid atropic acid tropic acid hyoscyamine 5-hydroxyindoleacetic acid indole-5-acetamide  -  + +  -  + +  -  _  -  INFILTRATION (minutes) 30 60 120 15  + +  + +  + +  —  —  —  -  mm  mm  mm  -  _  + +  + +  —  —  _  _  —  _  _  -  —  _  mm  mm  + + +  + + +  + + +  + + +  -  -  mm  mm  mm  +  mm  -  + +  -  + + +  mm  -  FIGURE 5, DIAGRAM OF TYPICAL AUTORADIOGRAM FROM INDOLEACETIC ACID-2-C ^ FEEDING  iso-propanol ammonium hydroxide water (20:1:2)  O 2 1  Note:  butanol, acetic acid, water (12:5:5) see following page f o r i d e n t i f i c a t i o n 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-C  14  COMPOUND ANTICIPATED  FEEDING EXPERIMENTS  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 tryptophan hyoscyamine phenethyl alcohol benzyl alcohol benzaldehyde l a c t i c acid  mm  :  mm  —  -  + +  +  mm  30.  FIGURE 6.  DIAGRAM OF TYPICAL AUTORADIOGRAM FROM UNIFORMLY RING LABELLED PHEMYLALAHINE-C  14  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 T R Y P T O P H A N - C 2 - I N D 0 L Y L ) - C  COMPOUND ANTICIPATED  + + +  tryptamine indole-3-aldehyde  -  acetonitrile  alanine skatole oC-phenyl-p-amino propionic atropic  FEEDING EXPERIMENT  LENGTH OF FEEDING (davs) 21  tryptophan  indole  14  acid  tropic acid  acid  + + +  hyoscyamine  -  indole-3-DL l a c t i c acid  +  indole-3-acetic  +  phenylalanine tryptophanol  acid  indole-3-propionic  acid  5-hydroxyindole a c e t i c a c i d  -  -  indole-3-acetamide  -  l a c t i c acid  mm  FIGURE 7.  DIAGRAM OF TYPICAL AUTORADIOGRAM FROM TRYPTOPHM-( 2 - I H D 0 L Y L ) - C  1 4  FEEDING  FIGURE 7.  COMPOUND NUMBER  (continued)  IDENTITY  1  tryptophan  2  tryptamine  3  indole-3-aldehyde  4  oC-pnenyl-p-amino propionic  5 6  atropic acid  7 8  indole-3-lactic  tropic acid indoleacetio  acid  acid  acid  34. TABLE VIII.  SOLVENT SYSTEMS EMPLOYED  SOLVENT SYSTEM NUMBER 1.  VERTICAL SOLVENT  iso-propanol  HORIZONTAL SOLVENT  200  butanol  120  ammonium hydroxide 10  acetic acid (glacial)  30  water  20  water  50  ethanol  16  n-propanol  5 5  ammonium hydroxide (28$  1  eucalptol  water  3  formic acid water  butanol  120  butanol  acetic acid (glacial)  30  IN sodium acetate  water  50  IN hydrochloric acid  sufficient to saturate 7 120 60  35.  TABLE IX.  CONFIRMATION OF THE IDENTIFICATION OF SELECTED COMPOUNDS FROM TABLES THREE TO SEVEN  TENTATIVE IDENTIFICATION FROM SOLVENT SYSTEM #1  <£-phenyl-,0 -amino propionic acid tropic acid hyoscyamine fumaric acid fumaric acid  l a c t i c acid l a c t i c acid phenylacetic acid tropic acid atropic acid  SOLVENT SYSTEM FOR RE-CHROMATOGRAPH  SOURCE OF THE COMPOUND  tryptophan-3-C14 seven day feeding ,14 indole acetic acid-2-C 120 minute i n f i l t r a t i o n 14 indole acetic acid-2-C 30 minute i n f i l t r a t i o n 14 phenylalanine-3-C seven day feeding uniformly ring labelled phenylalanine-C s i x day feeding tryptophan-3-C14 seven day feeding 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 twenty-one day feeding  14  2 2 3  2 2  2 2 2 2 2  CONFIRMATION  36.  TABLE X  DISTRIBUTION OF TBE C -LABEL IN THE DEGRADATION PRODUCTS OF TROPIC ACID DERIVED FROM THE TRYPTOPHAN-(2-INDOLYL) -C FEEDING 14  14  COMPOUND  carbon dioxide sjbyrene benzoic acid  Note:  f> YIELD OF THE REACTION  76.87 76.87 86.4  COUNTS PER MINUTE  52.5 65.0 0.0  COUNTS PER MINUTE CORRECTED TO 100% YIELD  68.25 84.5 0.0  The number of counts per minute was corrected to compensate for background radiation and for the efficiency of the apparatus. The scintillation f l u i d 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 manufactured by Pilot Chemicals, Inc.  37. DISCUSSION OF THE RESULTS The results shown i n 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* by participation of only the C* -carbon atom i n 4  4  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 i s incorporated into tropic acid, and that the label i s almost entirely on the carboxy carbon of tropic acid. The mechanism proposed by these workers i s 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 producing a 2-phenyl propionic acid structure. The net result of this reaction i s 26 the same as that found i n 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 structure. There i s no evidence to support the participation of a bicarbonate ion in this reaction in red clover, however, i t i s interesting to note that this type of shift has been found i n 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. 14 the fact that phenlyacetic acid-2-C  This i s shown by  i s an efficient precursor of tropic acid.  It i s possible therefore that bicarbonate produced from the degradation of tryptophan-3-C* , indoleacetic acid-2-C* , (by side chain oxidation), and 4  4  tryptophan-(2-indolyl)-C* , (by conversion of N-formy kynurenine to kynuren4  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* should 4  give rise to radioactive tropic acid with the activity concentrated largely 25 in the carboxy carbon. Leete and Louden have also used this material i n 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 i s led to the conclusion that since tryptophan14 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 i n table  number three. A l l 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, i s also a very labile compound metabolically.  Because of these two factors i t i s 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 i s indoleacetic acid. It i s 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 i n hibits the growth of root tissue. Quite likely the formation of indoleacetic acid i s the rate determining step i n the biogenesis of t ropic acid from tryp14  tophan. When indoleacetic acid-2-C  i s 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 i s quite clearly shown in table five.  In-  dole-3-aldehyde appears after three days of metabolism, <£ -phenyl-0 -amino propionic acid and atropic acid both appear on the sixth day, and tropic acid finally appears on the ninth day. figure one.  A l l of these are in the sequence shown i n  The results of the twenty-one day feeding experiment using tryp-  tophan- (2-indolyl)-C* shown in table seven, indicate that the same radio4  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 i n the removal of carbon atom number two of the indole nucleus 14  in the form of formate. Since i t i s this carbon which carries the C tryptophan-(2-indolyl)-C*  4 l  label in  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 i s synthesized within three days. The amount of isotope ut-  59. ilized for tropic acid production, i s estimated at approximately thirty per cent, as judged by the intensity of the spot on the autoradiogram.  It i s evident  that the ring of phenylalanine i s 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 without degradation of the phenyl ring.  i s incorporated  The mechanism for the biogenesis of  tropic acid from phenylalanine as shown by Underhill and Youngken Jr. i n figure two may well be correct i n essence.  Certainly the scheme i s difficult to prove  without the isolation of the actual enzymes involved since most of the intermediates would remain bound to the enzyme or would be unstable and difficult to isolate.  The only discrepancy i n this scheme i s the fact that the carbon  atom number one of phenylalanine i s shown to be given off as carbon dioxide. 25 The results of Leete and Louden  indicate that this i s not the case since  carbon atom number one of phenylalanine i s 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 i s indeed that of an intra-molecular shift where carbon atom number one of phenylalanine i s not lost but rather retained to form the carboxy carbon of tropic acid.  It i s  interesting to notice that phenylacetic acid, shown as an alternate precursor for tropic acid in figure two, i s not produced from phenylalanine-3-C the seventh day.  14  until  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 i n 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-C , indoleacetic acid-2-C , and uniformly ring labelled 14 14  phenylalanine-C  14  , are used as precursors. It would be expected that labelled  hyoscyamine would therefore be produced from tryptophan-3-C " 3  4  and tryptophan-  (2-indolyl)-C i f the feeding experiments were continued for a period of 14  twenty-four to twenty-seven days.  40. The results of degradation studies done on the tropic acid isolated from the tryptophan-(2-indolyl)-C' ' twenty-one day feeding experiment are 1  shown in table X.  4  These results show that tryptophan-(2-indolyl)-C  14  gave rise to tropic acid which i s 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^ would produce tropic acid labelled 4  specifically on carbon atom number three. The fact that carbon atom number one carried forty-four percent of the total C  found in the sample of tropic  14  acid i s not at a l l surprising. Judging from the intensity of the radioactive spots on the autoradiograms produced, i t can be said that approximately ninety percent of the tryptophan-(2-indolyl)-C was degraded via the formyl14  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 i n 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 i s not comparable, since i n their experiments using whole plants, the bicarb14 onate-C  would be fixed into sugars by photosynthesis. These sugars, es-  pecially the trioses and heptoses, serve as direct precursors for the aromatic amino acids. This therefore leads to uniform labelling of both phenylalanine and tryptophan and ultimately to the production of uniformly labelled 25 tropic acid. This i s the result obtained by Leete and Louden . The work done with isolated root tissue i n this laboratory did lead to quite a different result. Carbon atom number three of the tropic acid i s labelled by the scheme outlined by Goodeve i n 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" group produoed from the tryptophan-(2-indolyl) 14 14 -C . This free formyl-C molecule would participate i n the biogenesis of tropic acid from phenylacetic acid and i n so doing would specifically label carbon atom number one. It i s my suggestion that the tropic acid produced L4  41  from tryptophan-(2-indolyl)-C  i s 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 i s 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* offers direct proof for the indep4  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 i n Datura stramonium root tissue. Judging by the speed at which tropic acid i s produced and by the r e l ative amount of tropic acid produced, one would conclude that the pathway from phenylalanine i s the more important pathway of the two for the biogenesis of tropic acid.  One should bear i n 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 physiological pathways as the entire organism. The emphasis placed by isolated root tissue on the biogenesis of tropic acid from tryptophan i s certainly not great. This low emphasis may well be an artifact produced by the experimental method. The biosynthesis of indoleacetic 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 i s 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 i s one of the major metabolites of indoleacetic acid, and that the biogenesis of tropic acid i s the major mechanism for the degradation or detoxification of this plant hormone. In any event, the biogenesis of tropic acid from tryptophan definitely does occur i n isolated root tissue. Two pathways exist for the biogenesis of tropic acid in Datura stramonium  root tissue grown under aseptic conditions i n 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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