The Cactus Alkaloids. II. Biosynthesis of Hordenine and Mescaline in Lophophora Williamsii

The Cactus Alkaloids. II. Biosynthesis of Hordenine and Mescaline in Lophophora williamsii

J. L. McLAUGHLIN! AND A. G. PAUL (College of Pharmacy, The University of Michigan, Ann Arbor 48104)

The iV-methylated derivatives of tyramine, including N-methyltyramine and hordenine (N,iY-dimethyltyramine), have previously been identified as new alkaloids of the peyote cactus, Lo pho phora williamsii (Lem.) Coult. (14). The biogenesis of these alkaloids in barley rootlets, Hordeum vulgare L., has been studied extensively by Marion and his coworkers (5, 7, 8, 9, 12, 13,20). They have shown that 2-14C-phenylalanine, 2-14C-tyrosine, and I-l4C-tyramine are converted into iV-methyltyramine and hordenine. These workers have studied the origin of the methyl groups of these alkaloids using methyl 14C-methionine, methyl HC-choline, methyl !4C-betaine, and HC-formate. With the exception of choline, these compounds all served as precursors to the methyl groups of hordenine, and methionine methyl proved to be the most effective precursor. Mudd (16, 17) initiated enzyme studies on these methylations of tyramine. From barley rootlets, he has isolated a methionine-activating enzyme that converts methionine to S-adenosylmethionine, the actual methylating agent. Mann and Mudd (11) have succeeded in purifying the enzymes tyramine methylpherase from barley and tyramine and lY-methyltyramine methylpherase from millet, Panicum miliaceum L. It would appear from the above studies that these {3-phenethylamines arise in barley via decarboxylation of tyrosine to tyramine and subsequent step-v vise methylations to cY-methyltyramine and hordenine.However, Guggenheim (2) has noted the frequent natural occurrence of lV-methyltyrosine and has proposed that it and its hypothetical methylated derivative, 11',Jo-dimethyltyrosine, might be precursors of .Y-methyltyramine and hordenine in certain plants. To determine which of these two pathways might be used by L. ioilliamsii for the biosynthesis or hordenine, 2-14C-DL-tyrosine and I-HC-tyramine were tested as precursors to hordenine. A second goal of these investigations was to test the conversion of these two precursors to the hallucinogenic alkaloid, mescaline. Leete (6) has previously reported that tyrosine is a precursor to mescaline.Reti (19) has proposed that mescaline might be biosynthesized by conversion of tyrosine to dihydroxyphenyl- alanine (DOPA), followed by decarboxylation, another ring oxidation to nor- mescaline, and O-methylations to mescaline. An alternative to Reti's proposal is that tyrosine may be decarboxylated to tyramine and this, in turn, converted by oxidations and O-methylations to mescaline. The conversion of 2_14C-DL- tyrosine and I-HC-tyramine to mescaline would confirm Leete's results and sup- port this alternative proposa1. MATERIAL AND METHODS Plants.-Living specimens of L. williamsii,2 obtained from Mexico, were maintained at The University of Michigan Botanical Gardens. Thirty plants were transferred to a controlled environment chamber," watered every third day with distilled water, and maintained on a schedule of 12 hr light and 12 hr dark,

-Prcscnt address: School of Pharmacy,University of Missouri at Kansas City,Kansas City, Missouri 64110. 2Identifications were confirmed by Dr. E. U.Clover, Botany Department, The University of Michigan. 3Scherer Controlled Environment Lab Model No. CEL 512-37, Scherer-Gillett Co., Marshall, Michigan. 91 92 LLOYDIA [VOL. 30, No.1 with a light intensity at plant level of 3,000 ft-c. Both temperature and humidity were continuously recorded;' the temperature was maintained at 32° during the light period and 18° during the dark period, and the relative humidity was maintained at 80 and 100% during these respective periods. Administration of Radioactive Cornpounds.-Four large plants, 7-8 cm in diameter, which had been growing for 6 mo in the controlled environment lab were removed for administration of tracer compounds. These plants had not been watered for one week prior to their removal from the chamber. Each pot was broken to expose a portion of the large taproot. A total of 3.8 mg of 1-14C-tyramine HBr,5 with a specific activity of 5.75 me per mmole and a total activity of 0.10 me, was dissolved in 2.5 m1 of sterile water. Using a 2.5 ml sterile disposable syringe and a sterile 26 gauge, lYz in. hypodermic needle, 0.5 m1 portions of the solutions were injected into two sites in each of two plants.The first site was located at the junction between the green aerial portion and brown underground stem, and the second site was located approximately IYz in below this area in the large taproot. At the lower sites the needle was inserted into the plants until a resistance was sensed, indicating woody conductive tissue. The injections at the upper sites were made by inserting the needle approximately one inch into the center of the plants. After slowly injecting the solution, the needle was left in place for a few seconds to allow the solution to disperse. Similarly, 12 mg of 2-14C-DL-tyrosine,6with a specific activity of 1.4 me per mmole and a total activity of 0.10 me, was dissolved in 5 ml of sterile 0.05N HCl. Following the procedure described above, I-ml portions of this solution were injected into similar sites in each of two plants. After administration of the radioactive compounds, the plants were repotted, watered, and returned to the growth chamber. These four plants, as well as control plants, exhibited no harmful effects from the injections. Planchet Counting.-Solutions of the compounds to be counted were prepared by dissolving 2.5 mg in ethanol and diluting to 5 ml in a volumetric flask.One-ml samples of the solutions were pipetted onto duplicate 2.5 em planchets, and the ethanol was evaporated using a planchet spinner.Preliminary tests indicated that 500-,ug quantities of the compounds on this size planchet could be considered to be infinitely thin. A general purpose scaler" was used in connection with a flow of 0.95% isobutane in helium through a gas flow counter. 8 Planchets were prepared with 500-,ugquantities of a standard l4C-sucrose having an activity of 5.9 me per g. Each planchet was counted for six one-min periods. Background counts were subtracted and the net counts were averaged for duplicate samples.Corrections for counter efficiency and subsequent calculations of specific activities were made (1). Scintillation Counting.-8olutions of the compounds to be counted by scintillation were prepared in ethanol in the same concentration and manner as described above. The scintillation fluid was prepared according to the following formulation: PP09 7 g POPOp9 50 mg naphthalene 80 g dioxane, to 1000 ml Ten ml of the scintillation fluid was pipetted into a 27.5 mmX58 mrn, screw- cap, scintillation vial and 1 ml of the solution to be counted was added. After cooling at - 8° for at least two hrs the samples were counted in an Auto-

MARCH 1967] MCLAUGHLIN AND PAUL: CACTUS ALKALOIDS II 93

matic Tri-Carb Liquid Scintillation Spectrophotometer. 9 Solutions containing 500 jJ.-gof standard HC-sucrose were prepared in duplicate. Each sample was counted for three 10-min periods, background counts were subtracted, and the net counts were averaged for triplicate samples. Corrections for counter efficiency and subsequent calculations were made. The absence of quenching precluded the addition of internal standards. RESULTS Plants were removed from the controlled environment lab 4 weeks after administration of the radioactive compounds. The plants injected with tyrosine were arbitrarily called tyrosine-plants, and the plants injected with tyramine were arbitrarily called tyramine-plants. The tyrosine-plants and the tyramine-plants were extracted using the previously reported (14) purification method no. 2 to isolate nonphenolic (fraction C) and phenolic alkaloids (fraction E). M escaline.- The nonphenolic alkaloid extracts (fraction C) of the tyrosine- and tyramine-plants were treated identically to isolate, identify, and degrade mescaline HCI. Activities of the compounds were determined using the planchet method. The extract (fraction e) (14) was dissolved in a small amount of ethanol, filtered, and the filter paper was rinsed with ethanol.The combined filtrate and rinses totaled 7 ml. The solution was concentrated on a steam bath with the aid of a current of air to 2 ml, and I ml of 5% w/w HCI in ethanol was added. Upon addition of 5 ml of ethyl ether and cooling to - 23°, crystals of mescaline HCI were formed, filtered with suction, and rinsed with ethanol-ether, 1:9.After desiccation the yield, melting point, and activity of the crystals were determined. The mescaline Hel was then recrystallized by dissolving in 5 ml of ethanol, concentrating to 2 ml, adding 2 ml of ethyl ether, and cooling to -23°. The crystals were collected by filtering with suction and rinsed with ethanol-ether 1:9.The recrystallization was repeated. After each recrystallization the yield, melting point, and activity of the crystals were determined. After the second recrystallization a mixture melting point was determined with a sample of reference mescaline Hel. Ten mg of the recrystallized mescaline Hel was dissolved in 0.2 ml of water, and 0.2 ml of a saturated solution of picric acid in ethanol was added. The crystals of mescaline picrate were filtered with suction and rinsed with a few drops of ethanol. After desiccation the yield, activity, melting point, and mixture melting point were determined. Five mg of the recrystallized mescaline Hel was dissolved in 0.2 ml of IN Hel, and 0.1 ml of a solution, prepared by dissolving 1 g of chloroauric acid in 1 ml of water, was added. The needles of mescaline chloroaurate were filtered with suction and rinsed with 6 drops of water. After desiccation the yield, activity, melting point, and mixture melting point were determined. Sixty mg of the recrystallized mescaline HCI was dissolved in 5 ml of water and 0.1 mlof 10% ,TaOH. A total of 12.5 ml of 3% KMn04 was added, and the solution was boiled with stirring for thirty min. The volume was maintained by the addition of water. While still boiling, the excess KMn04 was destroyed by the addition of a few drops of ethanol. The solution was filtered with suction to remove the precipitated manganese dioxide. The filtrate was refiltered, acidified with 5 drops of concentrated HCI, and extracted four times with 25-ml portions of ethyl ether. Combined ethereal layers were filtered through anhydrous" aZS04 and evaporated to dryness on a steam bath with the aid of a current of air. The crystalline residue of trimethyoxybenzoic acid was dissolved in boiling water,

"Packard Instrument Co., Inc., La Grange, Illinois. 94 LLOYDIA [VOL. 30, No.1

TABLE 1. Mescaline H'Cl isolated from tyrosine-plants.

Isolated Reference Specific Compound Yield Compound Compound Mixture Reported activitv mg mp mp mp mp CPM/m"M ------_._------mescaline HCI (crude) 230 171-175° lS3° -- lS4° 5.74 x 105 (IS) mescaline HCI (recryst. 1X). 15S 174--177° IS3° -- -- 5.07 X 105 mescaline HCI (recryst.2X) .... S7 179-1S1° lS3° 180-182° -- 5 85 X 10' mescaline picrate ...... 16 219-221° 220-222° 219-222° 222° 5.27 x 105 (IS) mescaline chloroaurate. ... 11 138-140° 144-146° 14(}-143° 140-141° 5.23 X 10' (IS) trimethoxv- benzoic acid ...... 7.1 168-169° 168-169° 167-1690 1680 0 (3) filtered, and the container and filter paper were rinsed with hot water. Combined filtrate and rinses totaled 7 ml. The solution was condensed to 3 ml on a steam bath under a current of air. After cooling to 3° the crystals were filtered with suction, rinsed with 6 drops of cold water, and desiccated. The yield, activity, melting point, and mixture melting point were determined. Tables 1 and 2 summarize the data obtained from the mescaline HCI isolated from the tyrosine-plants and tyramine-plants. H ordenine=-« The phenolic alkaloid extracts (fraction E) (14) of the tyrosine- and tyramine-plants were treated identically to isolate, identify, and degrade hordenine. Activities of the compounds were determined using the liquid scintillation method. The phenolic alkaloids (fraction E) (14) were separated by chromatography using a silicic acid column. This column was developed with ethanol-chloroform, I:9, extruded, and segments eluted in a manner identical to that previously described under the TLC identification of tyramine (14). Eluants from the seg-

TABLE 2. Mescaline H'Cl isolated from tyramine-plants.

Isolated Reference Specific Compound Yield Compound Compound Mixture Reported activity mg mp mp mp mp CPM/Mm ------mescaline HCI (crude) .. 225 172-1760 1S3° -- 1840 3.00 x 106 (IS) mescaline HCI (recryst. 1X) . .... 144 177-1790 lS3° ------3.19 X 10· mescaline HCI (recryst. 2X) ...... 123 179-181° 1S3° IS(}-IS2° -- 3.43 X 10" mescaline 0 0 0 picrate...... 15 219-221 220-222° 220-222 222 3 26 x 10" (IS) mescaline 0 0 0 chloroaurat.e ...... S 142-144 144--14.6 143-146 140-141° 3.40 x 106 (IS) trime thoxy- benzoic acid. 8 168-1690 16S-169° 167-1690 1680 0.03 x lOG (3) ~'lARCH 1967) ;\ICLAUGHLIN AN!)PAUL: CACTUS ALKALOIDS II 95

ments were analyzed by TLC using solvent system A and tetrazotized benzidine reagent (14). Eluants richest in hordenine were combined in ethanol. Combined eluants were filtered into a 2 X 6 cm microsublimator, and the solvent was evaporated under a current of air. The residue was sublimed at 90° under the vacuum of a water pump, and crystals of sublimed hordenine were collected periodically over a number of hours. After desiccation the yield, melting point, and activity of the hordenine were determined. The sublimate was resublimed, and after desiccation the yield, melting point, activity, and mixture melting point were determined. Two hundred mg of inactive hordenine and the resublimed radioactive material were added to a 2X 10.5 em sublimator. To effect intimate mixing, the materials were dissolved in ethanol, and the ethanol was evaporated with a current of air. The residue was desiccated overnight and sublimed at 90° using a water pump. After desiccation the yield, activity, and melting point were determined. Ten mg of the active sublimate (diluted) was dissolved in 2 drops of ethanol and 2 ml of ethyl ether. One drop of 5% w/w HCI in ethanol was added, and the solution was cooled to -23°. The crystals of hordenine HCI were filtered with suction, rinsed with a few drops of ether, and desiccated. The yield, activity, and melting point were then determined. Ninety mg of the active hordenine (diluted), 5 ml of methanol, and 0.5 ml of methyl iodide were refluxed on a steam bath for 20 min. The solution was filtered and concentrated to 2 ml with the aid of a current of air. Five ml of ethyl ether was added, the solution was cooled to - 23°, and the crystals of hordenine methiodide were filtered with suction. Thecrystals were recrystallized by dissolving in 2 ml of hot methanol, adding 2 ml of ether, cooling to - 23°, and filtering with suction. The yield, activity, and melting point were determined. To liberate trimethylamine from this hordenine methiodide, a micro-Kjeldahl apparatus was arranged in a manner similar to the system described by Lintzel and Monasterio (10) for the liberation of trimethylamine from choline.The system employed a slight vacuum from a water pump that pulled air through a series of vessels consisting of the micro-Kjeldahl reaction flask, a trap containing 10 ml of 40% w/w NaOH, and two traps each containing 5 mlof 10% HCI. One hundred mg of the active hordenine methiodide was dissolved in 2 ml of water and 14 ml of 40% w/w NaOH. The solution was heated in the micro-Kjeldahl flask with a burner, and 15 ml of a saturated KMn04 solution was slowly added as the solution gently boiled. After 15 min heat was discontinued, but the water pump was allowed to pull air through the system for an additional 15 min. The system was dismantled, and the contents of the acid traps were combined and evaporated to dryness on a steam bath under a current of air. The white crystalline residue of trimethylamine HCI was dissolved in 2 ml of ethanol, filtered, and concentrated to 1 mi. To this solution was added 1 ml of a filtered solution prepared by dissolving 1 g of chloroplatinic acid in 5 ml of ethanol. The precipitated trimethylamine chloroplatinate was cooled to _23°, filtered "lith suction, and desiccated. The yield, activity, melting point, and mixture melting point were determined. The active hordenine (diluted) was acetylated and oxidized to p-acetoxybenzoic acid by slight modification of the method described by Leete, et al. (7).Ninety mg of the compound was acetylated by refluxing with 1 ml of acetic anhydride for three hours. Ten ml of water was added, and the solution was neutralized to pH 6 with K2C03. Twenty-five ml of 3% aqueous KMn04 was added, and the solution was heated for 15 min at 65°. A few drops of ethanol were added to the warm solution to destroy the excess KMn04, and the manganese dioxide was removed by suction filtration. The filtrate was refiltered, acidified with a few drops of concentrated hydrochloric acid, and extracted three times with 50-ml portions of ethyl ether. The combined ether extracts were filtered through anhydrous 96 LLOYDIA [VOL. 30, NO.1

TABLE 3. Hordenine isolated from tyrosine-plants.

Isolated Reference Specific Compound Yield Compound Compound Mixture Reported Activity mg mp mp mp mp CPM/mM ------1------hordenine I (crude) ...... 23 100--110° 116-118° -- 117-118° 54 93 x 105 i (15) hordenine (resublimed)...... - 13 105-113° 116-118° 110-115° -- 56.46 x 105 hordenine + carrier. 211 116-117° 116-118° 116-118° --- 4.30 x 105 hordenine HCI...... 8 180° 179-180° 179-180° 177° 4.24 x 105 (15) hordenine methiodide .. 152 230-232° 232-234° 232-234° 233-234° 4.'!9x105 (4) trimethylamine chloropia tina te. 33 223° 223° 223° 231° 0.08 x 105 decomp decomp decomp decomp (7) p-acetoxybenzoic acid...... 31 189-191° 191-193 ° 189-190° 188-189° 0.07 x 105 193.5-194° (7)

Na2S04 and evaporated to dryness. The residue was dissolved in boiling water, filtered, condensed to 5 rnl, and cooled to 30. The crystals of p-acetoxybenzoic acid were filtered with suction and desiccated, and the yield, activity, melting point, and mixture melting point were determined. Tables ~1and 4 summarize the data obtained from hordenine isolated from the tyrosine-plants and the tyramine-plants.

TABLE 4. Hordenine isolated from tyramine-plants.

Isolated Reference Specific Compound Yield Compound Compound Mixture Reported Activity mg mp mp mp mp CPM/mM ------hordenine (crude) . ... 41 112-117° 116-118° -- 117-118° 71.10 x 105 (15) hordenine (resublimed) . .. 33 115-117° 116-118° llG-11r -- 72 72 x 105 hordenine + carrier. 221 116-118° 116-118° 116-118° -- 9.88 X 105 hordenine HCI...... 6 178-180° 179-180° 179-180° 177° 9 12 x 105 (15) hordenine methiodide. 141 229-231° 232-234° 230-232° 233--234° 10.14 x 105 (4) trimethylamine chloroplatinate. . , .. 29 222° 223° 221° 231° 0.04 x 10' decomp decomp decomp decomp (7) p-acetoxybenzoic acid ...... 34 189-190° 191-193° 190-191° 188-189° 0.02 x 105 193.5-194° (7) :lIARCH 1967] MCLAUGHLIN AND PAUL: CACTUS ALKALOIDS II 97 DISCUSSION AND CONCLUSIONS M escaline.-Mescaline HCI from the tyramine-plants and tyrosine-plants was isolated, identified, counted, and degraded as follows. The nonphenolic alkaloids (fraction C) were dissolved in ethanol, treated with HCI, and the solution mixed with ethyl ether. The precipitated mescaline HCI was collected and recrystallized twice from ethanol-ether. The picrate and chloroaurate derivatives were prepared from the recrystallized product. Analyses of samples of the recrystallized mescaline HCI by the planchet method indicated a constant specific activity, and the two derivatives exhibited this same specific activity (tables 1 and 2). The recrystallized mescaline HCI was oxidized with KMn04 to inactive trimethoxv- benzoic acid (fig. 1). The degradations showed that greater than 99 per cent of the activity of mescaline from either precursor resided in the a-carbon atom.

PIG. I DEGRADATION OF MESCALINE

CH30DCOOH CH30V CH30 TRIMETHOXY- BENZOl C ACID

• INDICATES CI~

Of the total activity of L-tyrosine administered to the plants, 0.64 per cent was recovered in the isolated mescaline HCI; and of the total activity of the tyramine administered, l.53 per cent was recovered in the mescaline HCl. These results indicate that tyramine may be a more direct precursor of mescaline than is tyrosine. Since Leete has previously reported that tyrosine is a precursor of mescaline (6), these data confirm Leete's results, and, in addition, suggest that tyrosine is converted to tyramine, which in turn is converted to mescaline by a series of unknown steps. Assuming that only one pathway exists for the biosynthesis of mescaline and assuming that tyramine, which is present in trace amounts in the plant (14), is the natural substrate for the enzyme causing meta oxidation of the ring, this finding precludes alternative pathways involving oxidations and methoxylations of the tyrosine ring prior to decarboxylations. It is then conceivable that the next step in this pathway would involve m-hydrox- ytyramine (DOPAmine) (fig. 2). Since these results indicate that DOPA may not be involved in mescaline biosynthesis as Reti proposes (19), investigations are now underwav to test the conversion of both DOPA and DOPAmine to mescaline. Hordenine~-Hordenine from both the tyramine-plants and the tyrosine-plants was isolated, diluted with carrier, identified, counted, and degraded as follows. The phenolic alkaloids (fraction E) were separated by chromatography using a column of silicic acid developed with ethanol-chloroform, 1:9. Segments of the

FIG.2 PROPOSED BIOSYNTHESIS OF MESCALINE

COOH I• HOm "- NH2 TYROSINE 1 CH:30~. HO~. I •.. ------"- I NH CH 0" NH2 HO 2 :3CH 0 DOPAMINE :3 M ESCALIN E

• INDICATES (14 98 LLOYDIA [VOL. 30, No.1 column were removed and eluted with ethanol. The eluants were assayed by TLC, and those richest in hordenine were combined and subjected to sublimation. The sublimed hordenine was resublimed, mixed with carrier, and the mixture submitted to sublimation. Portions of the final sublimate were used to prepare the hvdrochloride and methiodide. The activities of each of the sublimates and of the two derivatives were analyzed using a liquid scintillation method. The analyses revealed that resublimation of the crude hordenine slightly increased the specific activity, and that hordenine plus carrier, hordenine HCI, and hordenine methiodide possessed nearly identical specific activities (tables 3 and 4). Tri- methylamine was liberated from the hordenine methiodide by boiling with basic KMn04. The gas was trapped in dilute HCI and the hydrochloride was converted into the chloroplatinate. A portion of the hordenine was acetylated with acetic anhydride and oxidized to p-acetoxybenzoic acid with KMn04 (fig. 3).

nc. a DEGRADATIONS or HORDENINE

P-ACETOXY- BENZOIC ACID

Analyses of the specific activities of the trimethylamine chloroplatinate and the p-acetoxybenzoic acid showed that more than 96 per cent of the activity of hordenine, derived from either precursor, resided in the a-carbon atom. Of the total activity of i.-tyrosine administered to the plants, 0.93 per cent was recovered in the hordenine; and of the total activity of tyramine administered, 0.99% was recovered in the hordenine. 'While these values are similar, they indicate that tyramine may be slightly better than tyrosine as a precursor of hordenine. Assuming that hordenine in L. williamsii is synthesized by only one pathway, the conversion of tyramine to hordenine precludes the derivation of hordenine from N,N-dimethyltyrosine as hypothesized by Guggenheim (2). Furthermore, the conversion of both tyrosine and tyramine to hordenine and the presence of N-methyltyramine and tyramine (14) indicate that the biosynthesis of hordenine in peyote is similar to the biosynthesis of hordenine in barley rootlets. SUM 1ARY Following administration of a-14C-tyramine and a-HC-tyrosine to plants of L. williamsii, radioactive mescaline HCl was isolated from the nonphenolic alkaloidal fractions and radioactive hordenine was isolated from the phenolic alkaloidal fractions. The active hordenine was diluted with inactive carrier. Derivatives of the alkaloids retained the specific activities. Degradation demonstrated that the activities resided in the predicted a-carbon atoms. These data indicate that tyrosine and tyramine can be precursors of mescaline and hordenine in L. williamsii. MARCH 19U7] MCLAUGHLIN AND PAUL: CACTUS ALIC\LOlDS 11 .99

ACKNOWLEDGMENTS This investigation was supported, in part, by fellowship GF~14,U94 from the Institute of General Medical Science, National Institutes of Health, U. S. Public Health Service, Bethesda, Maryland. The authors express gratitude to Dr. A: M. Mattocks, Mr. R. M. Kudla, and Mr. S. C. Mehta, for their assistance in the counting of radioactive samples. . . Received 19 May 1966. LITERATURE CITED 1. Chase, G. D. and J. L. Rabinowitz. 1964. Principles of radioisotope methodology. Burgess, Minneapolis, pp. 20~60, 187~I80, and 192~207. 2. Guggenheim, M. 1940. Die Biogenen Amine, 3rd cd, S. Karger, Basel, pp. 524~546. 3. Heffter, A. 1901. Ueber Cacteenalkaloide. IV. Mitteilung. Chem, Bel'. 34: 3004-3015. 4. Kirkwood, S. and L. Marion. 1950. The biogenesis of alkaloids. L The isolation of N-mcthyltyramine from barley. J. Am. Chem. Soc. 72: 2522-2524. 5. Kirkwood, S. and L. Marion. 1951. The biogenesis of alkaloids. II. The origin of the methyl groups of hordenine and choline. Can. J. Cliem. 29: 30-56. n. Leete, E. .1\J59. Biogenesis of mescaline. Chcm. Ind. (London) 1959:604. 7. Leete, E., S. Kirkwood and L. Marion. 1952. The biogenesis of alkaloids. VI.· The formation of hordenine and Nvmcthvltyraminc from tyramine in barley. Can. J. Chem. 30: 749-760. 8. Leete, E. and L. Marion. 19.53. The biogenesis of alkaloids. VII. The formation .of hordenine and N-methyltyramine from tyrosine in barley. Can. J. Chern. 31: 126c-128. n. Leete, E. and L. Marion. )954. The biogenesis of alkaloids. X. The origin of the .N- methyl groups of the alkaloids of barley. Can. J. Chern, 32: 64G-G4(J. 10. Lintzel, W. and G. Monasterio. 1931. Eine Mot hodc zur Mikrobcst.irnmung des Lecithins irn Blute und Plasma. Biocheni. Zeit. 241: 273-27\). 11. Mann, J. D. and S. H. Mudd. 1(JG3. Alkaloids and plant metabolism. IV. The tyramine mcthylphorasc of bnrlcv roots. J. Bioi. Chern; 238: :1R 1-385. 12. Massicot, J. and L. Marion. 1957. Biogenesis of alkaloids. XVI Ij. The formation of hordenine from phenylalanine in barley. Can. J. Chern, 35: 1-4. . 13. Matchett, T. J., L. Marion and S. Kirkwood. 1953. The biogenesis of alkaloids. VIII. The role of methionine in the formation of the N -mcthyl groups of the alkaloid hordenine. Can. J. Chem. 31: 488-4\J2. . 14. McLaughlin, J. L. and A. G. Paul. 1966. The cactus alkaloids: I. Identification of N-methylated tyramine derivatives in Lopho phora icillianisii. Lloydia. 29: 315~327. 15. Merck and Co., Inc. 1960. The Mcrcl: Index, 7th cd, Merck and Co., Inc.,. Rahway, N. J. p.254. 16. Mudd, S. H. 19()0. S-Adcnosylmethionine ·formation by barley extracts. Biochem. Bioph.ys. Acta 3.8: 354-355. ,.' 1.7. Mudd, S. H. 1960. S-Adenosylmethionine requirement for plant transmethylations. Biochcm. Biopliys. Acta 37: 164-1G5. 18. Reti, L. 1953. {J-Pbcncthylamines. In Manske, R. H. F. and H. L. Holmes. The alkaloids, chemistry and Physiology, vol. 3. Academic Press, New York. pp. 313-338. 19. Reti, L. 1950. Cactus alkaloids and some related compounds. Fortsch. Chem. Org, Naturstofl e 6: 242-289. 20. Sribney, S~ and S. Kirkwood. 1954. The role of betaine. in plant methylations. Can J. Chem, 32: 918-20.