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EH}, THESIS O ' lg, L [13 R A R Y Michigrm State University PATHWAYS FOR CWMIW 01’ T33 BETA CAI-{EON 0? 5mm TO m H-Mfim GROUP 0? NICOTINE By Stanley Gnrscn Krano A THESIS Submittad ta the Collage of Sciance and Arta Michigan atate university of Agriculture and Applied Science in garbial fulfillment 6f tha reguiremants for tha degree of mst OF 53 r: :13: Department of Chemistry 1958 14a~!-b”f gx'XI7J ACKNGELEDGMENTS The author extends his sincerest thanks to Dr. R. U. Byerrum whoae guidance and friandly assistance made this work possible. ?hanka are also due othar mambera of the Chflmistry Department for their willing advice. The author also expresses his apprecittian to the Department or Chemiatry and the National Institutes of aanlth for their financial aid. .QOQOOO¢OQGO 11 VITA The author was born February 16, 1937 in New York City. as “tended Dawn: Clinton High School. in the Bronx and them want: on to the City College of lieu York. The Bachelor of Science Degree flan conferred in m 01’ 1957. Tbs author entered Marxism State University in the Fall of 1957. Re served first as a Graduate Teaching Assistant and than u a. Special Graduate Research Assistant. W 111 PATHWAYS rm CONVERSION 01? m BETA CARBOH W 33mm: TO THE n-mmm. anon? W HICOTIKE BY Stanley Gamma lira-1119 AN "Ti’JLCT Submitted to.thn Colic a a: Science undertn Nic‘higan State Univera ty of Agriculture and Applied Scienca in partial fulfillment of the requirements for the degree of EASTER OF SCIENCE Department of Chemiatry Year 1958 Approved ___. X) (f. p ._ , w": ABSERACT Previous roseurcn had shown that coveral lubstlnces could.tunction an notabolic precursors of tho Homethyi group in nicotine. This study was undertaken in an nttcmpt to olucidtto u pathway by which the batsmbon or DL~aerinc is used for nynthccia or Ninethyl groups in.nicotina. The pathways investigated were (a) incorporation via formalde- hyde or on active i~carbon unit at the oxidation state or rammenydo, (b) by way or tomato or an mun l-carbon unit at tho oxidation state of tonmats, or (c) through transfer to tho methyl group ot’mcthionino. fl Tobacco plants were rod DL-serinc-3wcla alone and in combination with relatively large quantities of non- radioactive fornaldehydo, sodium formats and Dmeethionine, each in 5 separate experiment. Nicotine was isolated‘ as the dipicrato, in each case and its radioactivity determined. By comparing tho radioactivitiec of the nicotine obtained in each experiment it was shown that formaldehydc or an active l~ccrbon unit at the oxidation state of formaldehydo probably was an intermediate for the conversion or the beta carbon of cerino to the Namethyl group of nicotine. Formate or a i-carbon unit at the oxidation state of formoto was ruled out as an important intermediate. Tho results for'mothionino wero erratic and no final conclusion regarding its role as an intermediate for methyl group synthesis from corine's beta carbon was reached. INTRODUCTION. 0 i 0 TABLE OF CONTENTS 0 EXPERIHEITALAAHDJRESULTS. Growth and Pnporntion of tho non“. Administration and Upton: ot'zxporilontal compounds . Purity of tho DLnIortnno3- Isolation and Purificltion of Nicotine Results DISCUSSION . SUHHARY . o REFEREHCEs . APPENDIX . . O O O O O 0 vii O O O cit; ' O t O O G IQ. P880 ‘OMWS’R’H 12 17 18 TABLE II III LIST OF TABLES Paco Composition of the Stock fintriont Solution. . 5 Radioactivity of tho Nicotine Dipicroto. . . lo emporium or the Incorporation of the Beta Carbon 01' Sarina in Nicotine Depending on ComputinsCompomdPrcoent . . . . . . . 11 viii uncommon I HTR ODU‘CTI OH min: the put decade attain on maturation reaction: in plants hon boon extensively innotintod. tortioularly the smtheoio or the lonethyl group of the “unification, in tattoos plintax not rewind I good cm or attention. Report: by '31ch and semi oo- workers (N?) wound that the methyl group or uthioninc. the carbon of tomato, the toothy). groups of choline and (lyoim “tune. the alpha arm of ‘lycino. tho llpho cnrbm or glycolato. the bet: carbon or Dir-um. and tho carbon or romidohyoo could on function u ”mono precursor: of tho um‘tnyl mop in nicotino. ‘ It no. 90551121. to make emporium: of tn. oxtent of incorporation of tho above mentioned precursor: into nicotino. since the nourity and radioactivity no the can. throwhout. Tho methyl group: or mthionino, cholina, glycino botnim. tmmu carbon of uterine and cm Ilpmarbon or glycolnto nor. used for mthyl group synthetic to obout the some extent. Thu alpha carton of glycine was utilized about twice as much and tomato about one-tenth no much u the that five mentioned. The incorporation of formaldehyde was “out Moo that or the alpha carbon of glycine; home it would appear that formaldehyde is used for methyl group synthesis in nicmtlno ta-figzrsator'extanu thin any of tha othertprecursorn Itudlad. Rownvar the existanoo or tree faraaldehydo his navar been amnqnstruted in plants. An Iatlvo l-carban unit at the axidttlon Itatu of fonmuldahyda 1m been postulatad u the actual mtnylating agent (7). The presant study was undertaken in an attempt ta elucidate 5.9athway by which the bata earhon of DL~aér1ne in uaad for synthesis or qucthyl groups in nicotine. Three possible pathway: warn inwesttsatad. These Icro (a) incorporatlan via formaldehyde cr tha actlva l~carbon unit at the oxidation atate of tormaldahyfie, (b) by way at formats or a l—carbon unit at the axldatlcn state of formats, or (c) through transfer to tho mathyl of mathionine. The tachnique naed was to feed tobacco planta labalad serlne alane and in camblnation with a ralatlvely larga quantity of nan~radioactlve formaldehyde, farmata or'methlanlne, each in a ee§arate experimant. If any of the nan-radioacb1va aompounds were lntenmadiatea for the conversion or the beta carbon of aerlna to the Nwmethyl group at nicotine a decreaaa in the radioactivity of the nicotine laolated from the plants would b3 expected; alnce the radioactivity of tha precursor would be diluted by ths largn pool of non«radloactive intarmadiate. Howaver 1: any at the ncn~radicact1ve, compating Bubstancea were nut in tha-matabolic pathway of tha bata carban of serina no significant decraasa 1n nicotlna radioactivity wauld La 335993133er Gains this metabolic campetian technique the data obtained indicated that formaldehyde or a l-carbon unit at the oxidation state of formaldahyfia was an intermediate far the converaian of tha beta carban or aerine to the Khmethyl group of nicatine. Formate or a 1~carbon unit at the gxidation state at fafimata was rulea out as an tmportant intermadiata. The results fer methionine'ware erratic and no final conclusion regarding its role as an intermadiate for'mwthyl group syntheab tram eerine's beta cafiban was reachad. EXPERWAL AND RESULTS EXPERIMENTAL AND RESULTS growth and fimpamtion or the Plants The tobacco plants used in the experiments were Nicotlm Eating L. var. manna: I» strain with a high nicotine content. Thu plant. mm mm. in a green home. from seeds :31th in “mount-o («mentally «“1019 but Wed not). Thu tar. littered m a an Inn rod 3 nutrient sciatica tun. mm. Tho tantrum: solution was composed of 1 s. 3580307320, 1 x. [2190“, m 5.8 3. “(309243320 £11 diualved in 4 11mm of tap water. mu attaining a haunt. or 0.75 max»: tho punts m transplanted to u to :1" out: individual an m. The: plant: war. kept in tho men how until I mm: of 6 inches was reached. This took rrm two to than months. Upon manning tho haired height the plat. m 113mm from the vamiamto; their roots rinsed with up water and than clipped, with a minors, mar the sun. Thou punt: ware than plus“ in 125 ml. Erlcnmytr flak: containing 50 31. of an inorganic nutrient :olution'r: which was t 1:3 dilutian or a stock salution whose composi- tion 10 sham in Table I. TABLE I CCIIPOSITION OF THE STOCK RETRIEH? 8631.3?on mm— «ww-m-m—wwwwm-«mm-www ----~~-~ WW Hater ~ 1000 m1. ‘Magnesium sulfate 250 mg. Cancun nitrate 1 t. Antonina our». 250 as. Potaasim chloride 250 m5. Potassizm dihydrogan A-— M AAA Ferric chlorida 2 It. phosphate 250 u. The diluted stock nutzrient solution we oxygenated before the plants war-a placed in 1:. Each plan: had. a cotton plug wrapped aroma tha upper part or 1:: am and inserted in the neck 01' it. flask; The flasks were amend with black paper shields. Haw root watems vars purified to Genie]? on the” plant-3:: 3 process which took W 10 to 154 days. Any lass in volume of the original 50 ml. of diluted stock nutrient solution, during this films, was periodically made up nth distilled water. when the new mots had gram the plant: were red the matabalio precursors, of the fl-mthyl group of nicotine, to be described below. mm. tho feeding m6 afterwards, when the compounds fed were being metabolized, tm plants were kept in hoods supplied with artificial lighting. Two 36 inch 30 watt fluorescent tubes were kept lit 12 ham-a. of each day. iministratim find Uptake of Wrimntal 6033130!)de In order to manna significmtly the metabolic 99013 on!» nan-radioactive competing cmpomds, rod in canJmctxon with nL-aerlm-G-Cln, their cmoontrationa were made mtg:- than tho concentration of the radioactive marine. It and decided that tho non-radioactive competing compound in each experiment he made ten tins: grants: in concentration than the radioaouw vex-inc, on a molar basin. Each plant moaned L76 3: 10-3 @501. of radioactive norm. and 14.76 x 10-2 11301. of non-radioactive competing compoundr except the control experlmnnt 1n.uhioh the plants rooelvod only the n.75 x 10-3‘mmol. of radioactive aerlne. The non-radioactivv compounds rod along with radio- active scrim were DL-metnionlm, sodium tomato/Ind formaldehyde. Radioaotlvv Sarina alone was fbd no a control experiment. Each combination was made up of 1.9 m1. radioactive aerino, 1.0 ml. non-radioactive oompotlng compomd (0.0 ml in tho control experiment) , and nufflolent dilute aback nutrient solution to bring tho total voluma rod to each plant to 3.0 :1. Thu 3.0'31. of solution in each experiment war! placed in 125 ml. Erlenmeyer fladks which had blaok paper shields around them. The plant: which had generated new roots were placed in those flasks. Each plant was thou permitted to absorb the 3.0 ml. of solution. After these initial 3.0 m1. of solution were taken up, an additional 3.0 ml. oompoood entirely of dilute stock nutrient solution wan zlvon ouch plant. When this was absorbed, 50.m1. of dilute stock nutrlont solution was given each plant. The plants wore grom for com days after foodingfx coxmtinz the beginning of the first day as tho time when the original 3.0 ml. was fed. When the method or feeding outlined above had provioucly been used in thin laboratory for feeding radio- active acetate and radioactive tryptophan, uptake of the small volumes used was complete in two to three home (8). howevor in the present study complete uptake of the initial 3.0 ml. or solution took anywher- trcm 12 to 2!! hours. Ono further point or interest was that plants no the radioactive ccrinannon-radioactivo formaldehyde com- bination wilted during the seven day growing period following reading. 3% t: o; thg Dougegggg-rg‘“ The labeled aorinc was purchased from tho California Foundation for Biochemical Baccarat: (Loo Angelou, California). To to certain that no radioactive substance: other than Illin-iltcrinm-cu were present a sample was cowhranatographcd with pure non-radioactive DL-urino. Tho aolvent used was butanoiwaoctic acidwwator in a ratio of #3131. The radioactivity on tho chrmtoaramo was located with a Form ohrcmatosraph manner (Pom Scientific 60.. Emoton, Illinoia) coupled with Nuclear-Chicago model 1620A mtomctor and a nodal All Botcrlina Angus graphic mater. any one radioactive spot was found and its R: value corresponded to that of the non-radioactive cerinc. isolation and Purigiggtion ci' Nicotine At the end or the seven day growing period following the administration or the solutions described abovomicotino was isolated as tho dipicrato from tho planta. Tho plants worn removed from tho flack: and their roots rinccd with tap water and then blotted with I choococloth. The planta wore out into mall pieces and dried under a hoot lamp at about 80°C for six hours. The dry plant material was ground with a mortar and poctlo and placed in I. micro-Malachi flack with 20 per cont of its weight of calciuc hydroxide. Four drops of d. E. antifoam solution (0. E. Antii'oom 60. General Electric 60.. Schnectody, N. 2.) were addod and tho contonto arm. flask steam distilled. The distillate wan collected in a flask containing 5 ml. of 6 N hydrochloric acid. .Stcan distillation was continued until no more nicotine was present in tho diotillatef ac was ascertained with silico- txmgotic acid, Which forms a white precipitate with nicotino. Tho distillate contaning tho nicotino hydrochloride was evaporated to dryness in mg. The rocidual nicotine hydrochloride who purified by an azlotropic distillation from basic solution; again the distillate was caught in S‘ml. of 6 H hydrochloric acid (9). Tho purificd nicotine hydrochloride woo evoporatod to dmmoco in. W and the residue diacolvodin a small volmo of methanol and water. An excess of a saturated methanolic solution of pioric acid was sddcd and tho nicotine dipicrstc not allowed to precipitate. Tho precipitate was collected on a small sintcred glass filter and recrystallized from water. The melting point was 223°~22t°c.(mcomctec). ma reference records a valuo of 224°C. (10). Re to The radioactivity of the nicotine dipicrsto isolated in each experiment is presented in Table II. Tho specific acthities were corrected to zero sample thickness (soc Appendix for sample calculation). Tho specific activity of tho continstions rod in am I was 1.72 x. 107 com/moi of scrhne present. In.Runs II 1nd III tho activity of tho sorins in the mixtures {ad was slightly lower. Tho noximum specific activities of tho nicotine dipicrstes isolated in both.Runs II and III were corrected to corrcspond with the activity rod in Run 1. Tho isolated dipicrate was weighed, ground inrs mmsll agate mortar, and plated for counting on aluminum plsnchcts having an arcs of 2.83 sq. on. All radioactivity measurements word mode with a Nuclear-Chicago Model 192 x.scalcr (Hucloar Instruments and Chemical Corp., Chicago, Illinois) and a Traccrlsb Modol 50-16 proportional flow counter (Trccorlnb Inc., Boston, Host.). 10 fl} r [Lu 1‘ ‘1‘ 1 1 1 l gone m5: N.N m... m augmwawnflam ”3.0 flaw m4 3 N zuguww awe o N.” m.o m a mm.o m.mm H.m m m mamagom mm ”0 . Hm W. H # N I... wfinhwm ww o w.m .o m H bane w.»m m.H a m mcficoflzgma w.m m.w w.“ x m namaapmm mo 0 H. m m6 m « w.o m.ma q.~ a m mum «ohm urn w m 3qu03 a a w.a« m.H a a acaaam 48w you 393333 73v 7a. a .3033 053on @3393 353, 33mg wok coggngu «0 haubuuofi owhdoam mugowmun no of.» no .5” E H0 ragga.“ if MENMUHHHQ 535003 E B ngHadm HH gm. 11 TISSLE III CONPAfi SON OF WIS INCORPORATI N OF TH‘Z BITA C’”““1 OF SELRINE IN NICGI‘INE DEPEMIN ON CMPA ”TIM CWOUND P IFS EH3 (Specific Activity Nicotine Dipicrate in Con‘rol i) Specific Aétivity‘Nicotina‘bipicraia in rm. ’3: "L3 “1 MI {1‘ H {D :1 VJ % :3 I31 «.3 :2" £74 a. , m Rm: Sarina-Methionine Sarina-90mm Serim-Famldehyda “k + , ‘._). _._ 1 18 2 16 2 0.9 5 32 3 it 3 - 13 Ave.‘ 8:9 3:2 20:10 A. A A“ . _.. A _‘_. *'1_anuaa are tbs standard dcviationfl tram tha mean. 13186053103 12 DISCUSSION The nicotine dipicrnte isolated was radioactive; this indicated that in each or the experiments the beta carbon of DL-aerine was utilized for synthesis of fl-methyl groups. It was assumed that essentially all or the radioactivity of the nicotine was in the NAmethyl group. Thia had been indicated in previous experiments (7). An examination of Table III about that the ration presented therein are inversely proportional to the extent to which the beta carbon or aarine was converted to the methyl group. A large ratio would be indicative of a compound which competed to e large extent, in.methyl group synthesis, with eerine. Such a compound would be expected to not an an intermediate for the conversion of eerina’e betncerbon to the nicotine molecude. From Table II! it can be aeen that the largest ratio use obtained for the eerine-fonmnldehyde combinetion, the smallest for cerine-fornate and intermediate between thee. two In: the eerinednethionine combination. hence these data teen to indicate that for the conversion of eerina to methyl groupl'rorneldehyde probably in an inter- mediate. rennet. probably is not and methionine may or~may not be an intermediate. It in important to note that the range of values varied considerably in the eerino-methionine 13 ms. In fact 11‘ each or the three rms for the «mo uthionins combination were amused individuslly instead or as an average three different interpretions could he arrived at. Even tomato loweredths incorporation of urine into nicotine to some extent: thaw not very much when amazed to tomsldehyde. Thin can be explsined on the basis thnt {crate itself on be used to a slight extent for methyl group synthesis (1). However the boil: of formats probably does not arise tron the bets carbon of swim in tobacco Meets. The problem of methyl group synthesis has been investigated in animal metabolic. with regard to the relationships or the compounds studied in the present work. In 192w du Vignoaud (11) showed that the methyl group of methionine can be used for synthesis 01‘ methyl groups in choline. remote end fomsldehyde eleo can act as methyl precursors in choline (12): both being utilised to about the some extent. Jonecon and Kosher (13) demonstrated that the hate carbon or scrim and the carbon of formaldehyde could be used to synthesize labile methyl groups in rots. At the same time Weiesbsch, Elwyn end Sprinson (111) reported unnu- msulto for the alpha carbon of glycine and the bets carbon of urine; they also found that the cerboxyl . carbon of glycine was not used for methyl group synthesis. The alpha carbon of glycine was not used as much as the beta 11: carbon of urine. This eaae group or workers (15) also showed that the betacarbon of urine was not oxidised to {ornate} this is in acreemont with the result. obtained in the present study. It seems that in animal metabolism aethionine and glycine betaine aoommt for most of the nethyl group synthesis that takes place (16, 17). whereas in plants the alphaurbon of glycine and tomaldohyde appear to play the most important roles. mom:- 3g 5;. (7) moon-d mo fmaldehyde. the alpha carbon of glycine and the beta carbon of aerial light give rite to a emu active 1% Mt at the oxidation etate or romldehyde. hr: (18) and naliuk and Sakai (19) had previously poetulated the and thin. in their etudiee or aniaal aetaboliea. The biologically active l-oarbon unit appears to be a derivative of folio acid (20). The work of naliuk and Sakami (19) has given experimental smart for this. An atmpt has been me by m (21) to isolate radioactive folio acid m tobacco plants after feeding radioactive tori-aldehyde. The results were inconclusive but it appeared that a radio- active coupound res-amuse folio acid had been isolated. m following scheme is postulated for h-aethyl group synthesis in tobacco plants on the baais of the results reported in this etudy. Sarina (beta onrbon) Methionino Formate ° (methyl group) \Jrommehyda/ N—methyl group of Nicotine lothionine is visualized as yielding methyl groups either by direct tranamothylation or by oxidation to formaldehydo follouod by reauction. Serino has been postulated to give rise to tho methyl group or either nicotine or methionine by way of formaldehyde} although evidonoo for the latter pathway was inconclusive from the data presented here. Since the nature of the active 1-carbon unit is not known it has been excluded from tho oohomo. Ono furthor point that must be montioned is that Arnatoin (22) showed, in animal metabolism, that only the L~ioomor of Goring was used for methyllgroup synthesis. The work reported hora used DL-aerino: therefore, the difference of the two forms in plant metabolism ohould be invootigatod. Tho possibility of aerino'a beta carbon yielding methyl groups via transfer to mothionino (either directly ‘ or by an: or formldohydo) should be roinvoatisatod becauoo of the dubious results obtained. Other eXporimonts using different combinations of compounds (3.55. a series with radioactive formaldehyde and various non-radioactive competing canpomxds) might help to clarify the problem. 16 SUPMARY 1. 2. 17 SUHMnH Tobacco plants were fed DL-sorina—B‘Clh alono and in combination with relatively large quantities of non- rldioactivo formaldehyde, sodium format. and DL~ aothionino, each in I separate oxparimont. Nicotine was isolated, as the dipicrate. in each case and its radiooctivity determined. .33 oanpnrina tho radionctivitios of tho niootino obtainod in oach experiment it was shown that formal- dohydo or an active l—oarbon unit at tho oxidation state or formaldehydo probably was an intermediate for tho conversion of tho beta carbon of oorino to tho N~mothyl group or niootino. ‘Pormato or a l~onrbon unit it the oxidation state or tarmato was ruled out as an important intermediate. The result: for mothionino Hora erratic and no final conclusion regarding its role as an intermediate for methyl group synthesis from eorino'a beta carbon was roaohad. REFER? 3303333 10. 11. 12. 13. 113. 18 31.7”.” R‘INCES m 8. Ag, and 333m, R. U0, ’0 ”Am. Chem. soc... fl! 1523 (1952). Dewy, L. Jo, 31ng at U0, and Ball’ C. Dog J. Am. Chem. Soc..‘1§. 3997 (195“). warm, 3. 8.. and mag, 11. 3., .7. Biol. Chem... 3.9;, 637 (1953). Bate, C. 8.3 "Methyl Group Synthoaia in Plant metabolism, Ph.D. thesis, Michigan State University, 1955. Hamill, R. L., “Tho Role of the Alpha Carbon of'alycino in Nethylation Studios in Tobacco Plants,’ 14. 8. than, Michigan State Collego,1953. Dewey, L. 3., "Studies on the Biosynthesis of Nicotino and!I.iznin,“ Ph.D. thesis, Michigan State university. 195-4. Mormon, R. 3., Rineiez', R. 1..., Hamill R. In, and Ball. 0. 1).. J. Biol. Chem” 216, 331 (1955). Griffith, T. Jr., and Wu, P. 3., personalcammunication. Smith, c. 3., Ind. and Eng. Chem... 33, 251 (19142). Henry, '1‘. A... ”The Plant Alkaloids,” Biakiston 60., Philadelphia, Pa., 1959, p. 35. du‘Vigneaud,‘V., Chandler J. P. Conn, M., and Brown, a. 3., J. Biol. Chan... .133: 787 (19140). du‘Vigneaud,‘V., vorl , V. 0., and Wilson, J. 3., J. Am. cm. Soc... 13;, 2819 1950). Jonnson, 8., and Flasher, 3’. A... J. Am. Chem. 500-: 12: 3316 (1950). Weiasbach, A., Elwyn, D., and Sprinaon, D. 8.. J. Am. Chem. Soc., 13, 3316 (1950). 15. 16. 17. 18. 19. 22. 19 Elwyn, D... Heioobach, A... and Sprinaon, D. 8., J. Am. Chem. 800.. 11. 5509 (1951). du Vigneaud, V... Cohn, K... Chandler, J. P. Schmuck, J'. 11., and Simondn, 3., J. 21.01. Chem... 130, 625 (19:41). Stekol, J. I... Weiss, 8.. Smith, 9., and Weiss, K... J. Biol. cm... g}, 299 (1953). Berg, P” J. Biol. Chem... 325. 1115 (1953). Kieliuk, n. I... and Sakami, m. J. Am. Chem. Soc... .5, 1456 (195h). Rainer, A... "Biochemistry of The Amino Acids," Acadomio Press Inc... Row York, N. 2., 195?, p. 272. Brown, 0. 1)., "The Relationship of Folio Acid Cmpounda to one-carbon Metaboliam in Tobacco Plants,” 14.8. thesis, Kiohigan State University, 1958. mam. H. 3. Va, Biochem. :cp fig) 27 (1951). - APPENDIX APPENDIX The formula used in correcting the observed count to zero samplo thickness was Co 0 H ‘ m A” w - b when A, .. mum specific activity (countafiuinute/muimoie). co :- observed count (comta/minuto) H u noiocular weight of compomd w a weight of sample counted 1: a- fraction or maxim nativity ut tho ample thickness used (T)-obtainod from calf absorption OWCI Sample calculation: Ca ‘ 23(08 0.9.3., ‘1 . Bap6 m... H . 620 T ,‘ 12.2 Ego/Sq. “a. b ‘ 00m CHEMISTRY 11 A *1 r Date ue Demco-293