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 alka- loids of the peyote cactus, Lo pho phoRa Williamsii (Lem.) Coult. (14). The bio- genesis 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 com- pounds 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 main- tained 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 in- jected 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 scintil- lation 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- <Foxboro Humitex Recorder, The Foxboro Co., Foxboro, Massachusetts. "New England Nuclear Corp., Boston, Massachusetts. 6International Chemical and Nuclear Corp., City of Industry, California. 'Scaler model 146, Baird-Atomic, Inc., Cambridge, Massachusetts. 8Model FC-72A, Atomic Accessories, Inc., Valley Stream, New York. ,-- - _.- .. _------------------------------------- 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 ad- ministration 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 crys- tals 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.