AN ENVES‘FIGATEON OF THE EEQSYMHESES OF POLYTHEENYLS EN TAGETW ERECTA L. Tkesls §or {he Degree of DE. D. MICHIGAN STATE UNIVERSITY S. M. de Paul Palaszek, R. S. M. £965 THESls LIBRARY Michigan State University This is to certify that the thesis entitled An Investigation of the Biosynthesis of Polythienylo 1n Tagetes erecta. L. presented by S. M. de Paul Palaszek, R. S. M. has been accepted towards fulfillment of the requirements for 211.11;— degree in M ‘ / Major professor Date Novemng: 1]., 12§5 -»-—-—— __'—-.—- 0 0-169 a... lit .1-.. e . A l- . .v‘ ,2 v 1 ABSTRACT AN INVESTIGATION OF THE BIOSYNTHESIS OF POLYTHIENYLS IN TAGETES ERECTA L. by S. M. de Paul Palaszek, R. S. M. The intent of this investigation was to examine the role of acetogenesis in the formation of the thiophene ring of terthienyl and 5—(5-buten-1-ynyl)-2,2'-bithienyl in Tagetes erecta L., to discover the physiological precursor of the sulfur atom of the thiophene ring, to ascertain the relationship between the polythienyls of Tagetes, and to explore a possible route to the systematic degradation of terthienyl. Sodium sulfate—358, DL-glucose-UL-14C, DL-ornithine-Z- l4C, sodium malonate—Z-l4C, DL-methionine-2-14C, sodium pyruvate-5-14C, DL-cysteine—SSS, DL-cystine-1-14C, DL-serine— 3-14C, sodium pimelate—7-l4C and malonic-2—14C acid were examined for their relative efficiency as precursors to terthienyl in Tagetes erecta L. The radioisotope form was administered to the plants hydroponically. Terthienyl (and 5—(5-buten—1-ynyl)-2,2'-bithienyl in some experiments) was isolated from the roots at Specific times from the initial feeding, and its radioactivity was determined. S. M. de Paul Palaszek, R. S. M. The least dilution of the carbon-14 radioisotope in terthienyl occurred with DL-methionine-2-14C, malonic-2-14C acid, and pimelate-7-14C. Dilution factors of 1585, 1865. and 445, respectively, were calculated. Organic acids and malonyl CoA, therefore, have been shown to have a role in the biosynthesis of naturally occurring thiophenic compounds in Tagetes, and preformed polyacetylenes are not necessarily required in the synthesis. DL-Cysteine—ass has clearly been shown to be the pre- cursor to the sulfur atom in the polythienyls of Tagetes. A time dependent study of the incorporation of DL-cysteine-SSS into terthienyl and 5-(3-buten-1-ynyl)—2,2'-bithieny1 sug- gested that these polythienyls are independently metabolized at some point in their biosynthesis. Isolation of terthienyl after various metabolic inter- vals for sulfate-35$ and DL-methionine-2-14C indicated that 8 to 10-week-old plants were optimal for this work. Fungal and bacterial uptake of the radioisotope forms administered was shown to be insignificant. The absorption by root tissues, however, of DL-cysteine-sss and DL-cystine- 1-14C from aqueous solution has revealed a selectivity to an equilibrium Specie. The attempt to obtain terthienyldicarboxylic acid by a metal—hydrogen exchange reaction between terthienyl and S. M. de Paul Palaszek, R. S. M. n-butyllithium, formylation, and subsequent oxidation has led to a difficultly separable mixture of products. Acetylation of terthienyl has, on the other hand, provided a 57-47% yield of the monoacetylated derivative. Unreacted terthienyl, 25-51%, was recovered in the acetylation re- actions. Desulfurization of a crude sample of terthienyldicar- boxylic acid to a long chain acid was accomplished with slightly basic Raney nickel. The Beckman Monoxime degradation of long chain acids and the Barbier-Wieland procedure were examined. The latter gave 52 and 55% yields of the degrada- tion products. The Beckman Monoxime procedure was unsuccess- ful. This complete examination of possible precursors to the polythienyls in Tagetes decidedly indicates that an organic acid (pimelic acid) of high specific activity and known labeling pattern ought to be the precursor used in a degradation study of terthienyl. The scheme suggested by this work con- sists of conversion of S-acetylterthienyl to the carboxylic acid by the haloform reaction (already known), desulfuri- zation with slightly basic Raney nickel, and Barbier-Wieland degradation of the long chain acid. AN INVESTIGATION OF THE BIOSYNTHESIS OF POLYTHIENYLS IN TAGETES ERECTA L. BY - te“, .4 816M3(de Paul Palaszek, R. S. M. A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Chemistry 1965 ACKNOWLEDGEMENTS The author wishes to express her sincere appreciation to the Mother Provincial and Sisters of the Detroit Province, Religious Sisters of Mercy of the Union in the United States, for the opportunity and encouragement to undertake this study. She is eSpecially grateful to Professor Robert D. Schuetz for his direction and assistance during this investi- gation. Appreciation is extended to the following members of the Department of Chemistry for serving on her guidance committee: Professors C.-H. Brubaker, Jr., R. M. Herbst, K. G. Stone, and J. L. Dye, Associate Professor. Finally, the author would like to acknowledge with thanks the helpful discussions and equipment offered by C. H. Brubaker, Jr., Professor, Department of Chemistry; R. U. Byerrum, Dean, College of Natural Science; Floyd Challender, Plant Science Greenhouse; James Fleeker, Research Associate, Department of Biochemistry; D. D. Fossitt, Research Associate, Department of Biochemistry; A. Kivilaan, Assistant Professor, Department of Botany and Plant Pathology: T. B. Waggoner, M & T Chemicals, Inc., Ferndale; and, the graduate students, Department of Chemistry. This investigation was supported in part by Grant AT(11-1) 1034 from the Atomic Energy Commission. ii TABLE OF CONTENTS Page INTRODUCTION . . . . . . . . . . . . . . . . . . . . 1 EXPERIMENTAL AND RESULTS . . . . . . . . . . . . . . 25 Preparation of the Plants . . . . . . . . . . . 25 Administration of Labeled Compounds . . . . . . 25 Harvest of Roots and Extraction of Poly- thienyls . . . . . . . . . . . . . . . . . 27 Purification Procedures . . . . . . . . . . . . 51 Measurement and Determination of Concentration. 59 Measurement of Radioactivity. . . . . . . . . . 42 Dilution Factors. . . . . . . . . . . . . . . . 44 Synthesis of: 5-(2,2'-Bithenoyl)propionic Acid. . . . . . . . 46 5-Acetyl-2,2'-bithienyl . . . . . . . . . . . . 49 5-Acetyl-2,2';5',2"-terthienyl . . . . . . . . SO 2,2'-Bithienyl-5-carboxylic Acid. . . . . . . . 51 Desulfurization of 2,2'—Bithienyl-5-carboxylic Acid . . . . . . . . . . . . . . . . . . . 52 Synthesis of 5,5"-Terthienyldicarboxylic Acid. 53 Desulfurization of 5,5"-Terthienyldicarboxylic Acid . . . . . . . . . . . . . . . . . . . 58 Barbier-Wieland Degradation Sequence. . . . . 60 Permanganate Oxidation of 1, 1- -Diphenyldodec-1- ene. . . . . . . . . . . . . . . . . . . . 64 Attempted Beckman Monooxime Degradation Sequence . . . . . . . . . . . . . . . . . 64 DISCUSSION . . . . . . . . . . . . . . . . . . . . . 67 LITERATURE CITED . . . . . . . . . . . . . . . . . . 97 APPENDICES . . . . . . . . . . . . . . . . . . . . . 103 iii LIST OF TABLES TABLE 1. 9. 10. 11. 12. 15. 14. 15. 16. 17. 18. 19. Naturally Occurring Thiophenic Compounds Isolated 1947-1962 . . . . . . . . . . . . . Recently Isolated Thiophenes . . . . . . . . Recently Isolated Polythienyls . . . . . . . Proposed Biological Pathways to Terthienyl . Products of Hydrogen Sulfide Addition to Polyacetylenes . . . . . . . . . . . . . . . Biosynthetic Data for Terthienyl . . . . . . Some Thiophenic Natural Products and Related POlyacetYleneS O O O O O O O O O O O O O O O Thiophenic Compounds from Echinops sphaero- cephalus L. . . . . . . . . . . . . . . . . Growth Conditions. . . . . . . . . . . . . . Radioisotope Forms Administered. . . . . . . Study of Fungal and Bacterial Uptake . . . . Uptake of Radioactivity. . . . . . . . . . . Acetylation of Terthienyl. . . . . . . . . . Thin Layer Chromatography of Terthienyl. . . Identification of Biosynthetic Polythienyls. Autoradiogram Data . . . . . . . . . . . . . Double Dilution Method . . . . . . . . . . . Experiment 11: Barium Carbonate Counting. . Incorporation Data for Terthienyl. . . . . . iv Page U'ICNN 12 15 16 17 24 26 28 50 55 35 56 58 41 44 45 LIST OF TABLES - Continued TABLE 20. 21. 22. 23. 24. 25. 26. 27. Incorporation Data for 5—(5-Buten-1-ynyl)- 2’ 2".bithieny1 o o o o o o o o o o o o o o Esterification of Fatty Acids. . . . . . . Incorporation Data for Echinops Sphaero— cephalus L. . . . . . . . . . . . . . . . Variation in Biosynthetic Terthienyl . . . Percent of Radioactivity Recovered in Nutrient Solutions . . . . . . . . . . . . Incorporation of Sulfur-55 into Terthienyl DL-Cysteine-sSS Incorporation into PolyL thienyls . . . . . . . . . . . . . . . . . Fluorescent Components of Ethanol Extracts Page 46 60 71 75 79 82 85 89 FIGURE 1. 2. 5. 4. 10. 11. 12. LIST OF FIGURES Autoradiogram, Experiment 22 (D1) . . . . Autoradiogram, Experiment 25 (D1) . . . . Autoradiogram, Experiment 21 (D1) . . . . Infrared Spectrum propionic Acid. . Infrared Spectrum Infrared Spectrum terthienyl. . . . Infrared Spectrum Infrared Spectrum Infrared Spectrum canedioate. . . . Infrared Spectrum ene (neat). . . . of 5-(2,2‘—Bithenoyl)- of 5—Acetyl-2,2'-bithienyl of 5-Acetyl-2,2';5',2"- of the 404 mu Product . of the 568 mu Product . of Dimethyl Tetrade- of 1,1-Diphenyldodec-1- Time Dependent Study on Sulfur—55 . . . . Time Dependent Study on Carbon—14 . . . . vi Page 57 57 57 48 48 48 57 57 59 59 86 86 LI ST OF CHARTS CHART Page 1. Partial Acetyl CoA Flow Chart for the Plant Kingdom . . . . . . . . . . . . . . . . . . . 1O 2. Tritium Labeling Experiment with Echinops sphaerocephalus L. . . . . . . . . . . . . . 19 5. Frequency Distribution of Radioactivity . . . 52 4. Naturally Occurring Polyynes and Related Thio- phenic Compounds. . . . . . . . . . . . . . . 68 5. Biosynthetic Scheme A . . . . . . . . . . . . 75 6. Biosynthetic Scheme B . . . . . . . . . . . . 91 vii LIST OF APPENDICES APPENDIX Page 1. Source of Labeled Compounds . . . . . . . 104 2. Formulae for Counting Statistics and Errors (64,65). . . . . . . . - . . . . . 105 viii INTRODUCTION The divergence between plant and animal chemistry is reflected in the discovery of thiophenic compounds in the tissues of higher plants. Biotin, a tetrahydrothiophene, is the only known simple thiophene, I, derivative common to plant and animal tissues. The polythienyl, terthienyl, II*, discovered by Zechmeister and Sease (1) in 1947 in extracts of Tagetes erecta L., is the first representative of thiophenic natural products of higher plants. Of the forty-four known thiophenic compounds, Tables 1 to 5 and 7, sixteen are discoveries of the current year. All these compounds are the constituents of species in the Compositae Family except junipal, a fungal fl /\/\/\ I II Thiophene Terthienyl product. *2,2';5',2"-Terthieny1 is the nomenclature approved by the I.U.P.A.C. 1957 rules on Organic Nomenclature. Terthienyl is used throughout this work for simplicity and the unambiguity of the term in this context. snow IHHHmUcmam mmummme AHocmnum mania muomum mmuwmme .mcmxmsvomm.mmm /m\ /m\ m ma muooum mmummma Amcmuooomflvoem.dmm NmOanOMUIA/m\y. m MHHomwmasumm mchHm mm 3363 9568 Aocmxofimmmdmm mmoumomonmouA/m \w' loom mHOUOCH mm mflnmofluomz mmommooomxmmovmoumoomou m MHOUOCH mm mflumofluumz mmommooomxmonmovomou m muoooca mm mflumofluumz mmflmonmovomou m mummas> «m sasmnocmmsnno aem.mmm mmoooomoumo- -omoomm ummBm xm mmom ea muonwbcmum mammomuoo chmxmLVOdm mmoUI IOmOUmm AmdmcsmVMCHummflcsh m mflaooomo Aaocmnumvomm.mmm.mam omou nomoomm Aaev .m m .mmm mmflummm mpcmm coaumuomnfi muamsuwumnsm m mIAWVUWIIm .Ammv mmmauaama ooumHOmH mocsomsoo oflconnoflss mcfluusooo adamusomz .a magma Umscwucoo >> mumccflm Amcmuuvmoooo.o o we mcHHoucmm Amfiovmo.o Aocmxo£VAmmmvmmm.omN A/ 1%lmuanOMOI m mcoummuuoum .EooomHDUHcmom mw.a¢ Essmnucmmmuno mAmmoVmommomzoomoumomonmommou m mmommuo ow maumoauumz Esmomubcwawo Ha assocucmuox a0.0 Auocoovmmm.mmm.>mm mmoumomoumomoumoomou m AucmHm mHO£3vmumH©mu mm.aa mHsumunom mooo.o AuwnumVAmmmv.mmm mmumonmoomul IomomOuOmm 5 mumumflum Amonumvmmm mumumom AH mfiummm «000.0 Romanovmmm m0mmomoumoomou nomomouomm mumumflum ea mflummm $000.0 Amoroovxommv.0om.00m omomonmoomou nomomouomm mHHSMOmeHmEm Ha mflxoonosm 0.H Aumcumvamm.amm.mmm mmoumomoumoomo- -omoomm mUHOHomcmm Aumcumvnmm.mmm Ha mflunsxcom a0.0 .Aemmv.wam.e0m.0mm mmoumoomoomou nomoomm AR uanmBV Ajay .m m .mwm mmaummm huwucmso mbcmm Goapmuomnd mucosuwumnom m .m IAmyMWI. m musuosuum may mo mmcwnmoflns UmumHOmH waucmowm .m OHAmE mcmummuonuo om mamafimuud mmOOmOIIAWWVWWIOOOmm oomm mcmommuonum ow memflsmuu< mmOOmOIIAWWMWWIOOOmm . OE .m mumomsuw as mflsonocexmcmu50¢00.0 Anonumvmom.0mm.xmmmv mmoooomonmoomo: m .A mflaflnoc we mHEonuce «00.0 , Hozuovmmm mmoooomoumoomo: nomm MCMHOH>OUSH O we mflmflsmuua No.0 AuoaomVAmemv.Rmm.xmam0 ///umo- m 0 he unmflmzv 1150 .m m .mmm mmflommm wuflucmso wocmm COHumHomQ¢ mucmsuflumnsm UOSCHUCOO I N manna w 'c n In . . or. E” cabs-.V.IAV\ t...- nUN;§-.-. .. u. n.. am~t#-..-..~ .tfiu...— .5... ~... ~ ~ . .I‘V— .v..at..~.-v.. . -. .Zl// \\ // \. mm Aucmam maon3v moumccoo mcmoflm 0a00.0 Anonuovmmm.mmm.emm mmomoumou :mouomm AucmHm maon3v mm mmpH0flH£mp mchHm Nooo.o mmuumOOmOI Iofimovmm AucmHm maon3v mm mmUHOHHrme mcoonm 0000.0 mmoumoomon Immooooomm me MDDCHE mwummme www.mwm HommofimovaUmUI m we ouSCHE moummms we muumum mmummme mmo.o mmUOUONmUNmUOMOI m we muSCHE mmummme www.mmm.mem mONmONmOOMOI m we muomnm mmummme mao.o Aumnumvmwm.amm Sm muscHE moummMB mo.o awm.amm mmOHmOOmOI m we m mm museums“ mmummma ao.o Amcmxmnvomm A/ \VI m AR oneness 1150 .m m .mmm wmflommm >uwucmso mocmm coaumuomnd mucmsuflumnsm m .mlfl/ \VIIIA/ \Vlm Unduosuum on» m0 maxcmflnumaom bmumHomH Saucmomm .m. magma The biosynthetic origin and mode of formation of the thiophene ring in natural products have not been definitively established. Several biological pathways to terthienyl have been proposed; schemes A to D (Table 4) are based on con- siderations of biogenetic* evidence and practical laboratory syntheses. Schemes E and F are the results of biosynthetic* investigations with Tagetes erecta L. (2,5), Chrysanthemum segetum (4), and Echinops sphaerocephalus (5). A favored origin of the carbon skeleton of the thiophenic compounds from higher plants is a long chain polyacetylene. The biogenetic support of this hypothesis cites the similarity in structure and source between terthienyl and polyacetylenes. Since the thiophenic natural products reported except junipal (6) are constituents of one taxonomical group of plants, Compositae, identical biosynthetic pathways among the thiophenic compounds appear to be operative. All natural thiophenic compounds possess an unbranched chain of ten to thirteen carbon atoms and are generally isolable from plant root tissue. The polyacetylenes, compounds containing conjugated triple bonds, are widely distributed in the Compositae Family (7,8,9,10). Their formulae range from eight to * Biogenetic denotes an evolutionary development from pre-existing biological material. Biosynthetic is the term used in referring to the production of a chemical compound by a living organism utilizing synthesis or degradation. m ATII. «a.¢ .mIAWIww .momomoumoumomm mmmmo + mmxomovm _ m m mm m omm\_ V 0000” V m mmz mm m m. a ma mmoumoomo / \ / \ 0mm + m Aomovomom 0 m csocxcollv mIIAmWVW IIIIIv momoomom o C m C m Aoommov.lAW|MW m xomovomom «a mooommomxoommovomoomom mooommovxoommovooomm m ma mamacmEuoucH c30chD mmm + .mofiomovm < mocmummmm mmumwbmfiumucH HOmHsomHm Ummomoum mfimnum axcmflnuume ou m>m3£umm HMUHmoHOHm Ummomoum .¢ magma eighteen carbon atoms in a continuous chain with some preference for chains of ten and thirteen carbon atoms. The polyyne III occurs abundantly among one-hundred forty genera of the Compositae Family,* and IV is typical in ten of twelve tribes of this Family (11,12). Derivatives of acids, methyl esters and isobutylamides, are the simpler acetylene compounds found in the root and aerial parts of the plants. H3C(CEC)5CH=CH2 H3CCH=CH(CEC)4CH=CH2 III IV The well—known instability of compounds with a system of extended conjugated triple bonds (15) is the basis of Sorensen's (14) proposed biological pathway,Scheme B (Table 4). This pathway assumes that the dehydration and the dehydrogenation of two oxo-functions of a polyketone pro- duces an acetylenic ketone. A monothienyl derivative is formed by addition of hydrogen sulfide across the conjugated triple bonds with cyclization. Repetition of this sequence of reactions yields terthienyl according to this Scheme, while the omission of the reaction with hydrogen sulfide produces the polyacetylenes of the Compositae Family. The discovery of 5—(5-buten-1-ynyl)-2,2'-bithienyl as a com- ponent of the root extract from Tagetes erecta in which *One classification of the Natural Order, Compositae, designates 806 genera for thirteen tribes (7). terthienyl had been isolated is taken as biogenetic support for the sequential formation of the thiophene ring (15). The structures of all known thiophenic natural products are suggestive of this mode of formation. The requirement for a polyketonic or polyacetylenic structure is ultimately supplied by acetyl CoA* in biological systems. Chart 1 summarizes the experimentally demonstrated and hypothesized pathways to polyacetylenes and polyketones. Nemotinic acid (V) and matricaria ester (VI) from fungi (16) and matricarianol (VII) from seedlings of Santalum acuminatum (10,17) show an alternate labeling pattern when grown in the presence of malonate-2-14C and acetate-1-14C respectively. Thus, these polyynes, as acetate-derived are established. HCECCECCH=CFCHCH(0H)CH2CH2C00H V H3CCH=CHCECCECCH=CHCOOCH3 VI H3CCECCECCECCH=CHCH20H VII Laboratory syntheses justifying the biogenetic argu- ments that the thiophene ring in biological systems could be formed by the addition of hydrogen sulfide to an acetylenic *Acetyl Co-enzyme A is an enzyme thiol ester of acetic acid. 10 Chart 1. Partial Acetyl CoA Flow Chart for the Plant Kingdom Polyacetylene$6L1g,47_JEnOl ] 6 Benzenoidsfififl.£9_Polypyrone<_ _6‘_,_4§ _Polyketone Orsellinic aci 49 9 Malonyl CoA Glucose 50,51 thruvate 50 cAcetyl CoA 52,55,54_9Acetoacetyl CoA 16,54,55,49 Polyacetylenesthe thienyl function. Jones (9) is notably alone in propOsing that the thio- phenic natural products may be the source of polyacetylenes in nature rather than metabolic products of polyacetylenes (Scheme C of Table 4). It is well—known that Raney nickel desulfurization of thiophene compounds is utilized synthetic- ally to obtain fatty acids of a desired length and with a specific carbon-14 labeling pattern (21). The first biosynthetic investigation of the origin of terthienyl in Tagetes erecta was a study of the incorpora- tion of sulfur and carbon radioisotopic forms (2,5). The data, Table 6, show a high incorporation of inorganic sulfate, low incorporation of methionine-2—14C and ~358, and negligible incorporation of acetate-1-14C and bisulfide-SSS. All feed- ings of likely precursors to the plants were by hydroponic Table 5. Products of Hydrogen Sulfide Addition to Poly- acetylenes Reactant Product Reference H3CCECCECCH3 ch-Q-CHg 19 C6H5CECCECC5H5 C5H5—/S\ -C6H5 19 C4H38' CECCZ—C SC4H3 C4H33- / s\ -SC4H3 19 H 3c ( CEC) 3CH=CHCOOH H3CCEC-© -CH=CHCOOH 1 9 H3C(C;—':C) 3CH20H H3CCEC-O-CH20H 19 C4H3$ (csc)3c1_1(on)c1r13 C4H38 CEC-Q-CH(OH)CH3 20 C6H5(CEC) 3C5H5 CsHs-Z/ S \5 "CEC C5H5 19 C3H5(CEC) 4C5H5 ceHs-g S\>-(CEC)2C6H5 19 15 Table 6. Biosynthetic Data for Terthienyl 'RadioisotOpe Incubation Period Dilution Form (days) Factor Bisulfide-aSS 1.5 oo Sulfate-35$ 2 294 Methionine-2-14C 2 1450 Methionine-358 1.5 4420 Acetate-1-14C 2 oo Acetate-1-l4c 10 55100 administration since stem feedings showed no incorporation even with inorganic sulfate. The tenuous suggestion that the thiophene rings of terthienyl arise from the cyclization of a four carbon unit, condensation of the cyclized unit with another carbon fragment, cyclization, and a repetition of these reactions is one reasonable interpretation of these data (Scheme E of Table 4). The intact methionine molecule is not likely to be the precursor to terthienyl since the labeled carbon and sulfur methionine are in- corporated into terthienyl to different degrees. Cystathionine is related to the sulfur containing amino acids and may be an alternative precursor in this Scheme. The value of this type of investigation of likely pre- cursors to any biological material is directly related to the value accorded to in vivo evidence as contrasted to 14 ig'yitgg and biogenetic arguments. Meinwald (22) recently employed a study of likely precursors to refute the sup- position that preformed terpenes are required for the bio- synthesis of citral and citronella in arthropods. The biosynthetic origin and mode of formation of the thiophene ring in natural products is still nebulous. The intent of the investigation described here was to investi— gate the role of acetogenesis in the formation of the thio- phene ring, to discover the physiological precursor of the sulfur atom ofxthe thiophene ring, and to ascertain the relationship of terthienyl to 5-(5-buten-1-yny1)-2,2'- bithienyl in the Specie Tagetes erecta. This plant is a favorable choice for this study Since historically it was the first plant in which terthienyl was discovered; it is a sturdy plant; and, it is easily grown from seed. Concurrently with this study, investigations reported by Bohlmann and his co-workers are creditable with bio- genetic and biosynthetic contributions to the mode of formation and origin of the thiophene ring. The intermediate between the biogenetically proposed conjugated triple bonds and a thiophenic structure is suggested by the addition of methyl mercaptan or its biological equivalent to the terminal triple bond of VIII (4). The polyacetylenes VIII and IX CeHSCOCECCECH C5H5COCECCH=CHSCH3 VIII IX 15 are isolable from Chrysanthemum segetum L. The former, labeled with carbon—14 at the oxo-carbon atom, was adminis- tered to Chrysanthemum segetum L. plants. The degradation of the sulfonyl derivative (96000 cpm/mM) of IX to benzoic acid (84000 cpm/mM) and confirms the transformation of VIII to IX in a biological system. Additional biogenetic evidence for the metabolic path- way in Scheme F, Table 4, is cited in Table 7. Table 7 is a compilation of polyacetylenes and comparable thiophenic materials present in the same genera. The tribe Anthemidea contains the genera Chrysanthemum and Anthemis. The tribe Helenieae contains the genus Tagetes; and thistles, for example, EchinOps Sphaerocephalus L., are part of the tribe Cynareae (7). A tedious separation by repeated column and vapor phase chromatography was reported by Bohlmann and co-workers (25) to yield twelve new thiophene compounds and two polyacetylenes from the same extract of roots. The structures and quantities of the compounds obtained from the roots of Echinops sphaero- cephalus L. plants are summarized in Table 8. The compounds are biogenetically related to a penta-yne, X. Compound X, although never isolated from this Specie, is found widely distributed in the Compositae Family. The results of an .13 vivo investigation with 1,2-tritium labeled X and Echinops sphaerocephalus L. is diagrammatically shown in Chart 2 (5). ) H3C(CEC)5CH=CH2 X 1000 .m moucmownomun Aemv mummas> m mHEmcuc¢ mom EoEmnucmm>u£O m .muoom mmOOOOmUHmUOmUmUHWUmUUmm .muoom mmUOOUmUHmUIA/ \QIOMUUmm 1000 .q mfluouocflu mmom 1990 mHEmnucd .m mumomsum mHEmnuc< m .muoom mmoooomoumom Aomovmouuwomm .muoom mmoooomoumoomoufl/ \V 6 Aaao flames AHSV samba .1 Esuuflnlooflomfl> Enuuflnlooflomfl> ESEmnucmmwunu O Edamnucmmhunu .mm>mmq A //umOmAOmOVOmm .mm>moq 0 II. Aawv EsmumHsoHCmom mmOOOO Aawv EsmomHoUHcmOM Esfimnucmmmucu O// Enamnucmmmuno m .muoom Imomxomovomm .muoom mAmmovmommomzoomAmonmovmmou o l / .\ muusom mamawumummaom wuusom m>Hum>fiHma mamSQOASB nonmamumum>aom UmumHmm Ucm muosvoum Housumz Uflcmcmoflns mEOm .m wfiflme 17 Table 8. Thiophenic Compounds from Echinops sphaerocephalus L. (25) Structure Column Quantity Fractiona (weight %) H3C(CEC)3—CH=CHCOOR 2 0.000085 H3C(CEC)3-(CH=CH)g-CH(OH)CH2CH20H 4 0.0085 H3C(CEC) 2-Z/ \>‘CECCH=CH2 :l. 0 .00041 s H3C(C_=_C)2-Z/ \X-CECC‘HFHZ 2 0.000085 3 o H3C(C.=_-C) 21/ \) -CECCH2CH20C0CH3 2 0.00025 5 H3C(CEC) 21/ \) -CECCHC1CH20C0CH3 2 0.0016 s H3C(CE)2 -(/ \X-CECCH(0COCH3)CH20COCH3 5 0.00016 H3C(CEC)2- -CECCHC1CH20H 5 0.0020 s H3C(CEC)2-Z/ \) -CECCH2CH20H 5 0.00041 9 H3C(CEC)2--CECCH(0H)CH20COCH3 4 0.00025 5 s / \ / \-CECCH(0H)CH20H 4 0.00062 S S b /\ /\ / \ 1 0.012 S S S aColumn fractions: petroleum ether (1), etroleum ether-10% ethyl ether (2), petroleum ether- 50 ethyl ether (5), and ethyl ether-10% methanol (4). bAlso from Tagetes erecta and/or minuta. 19 Chart 2. Tritium Labeling Experiment with Echinops sphaerocephalus L. H3C(CEC)5-CT=CTH 5.00 x 109CPM/mM @3c(czc)2-Z7 )B-CSCCT=CTH] s / \ / \-CECCT=CTH H3C(CEC)2-Z/ \B-CECCTCICTHOCOCHg s s s I 7.11 x 106 CPM/mM } : . I I :37 ‘7’ Z/ \LZ/ \B-COOH H3C(CEC)2-Z/ \X-CECQTCTH s s s o/ // o CPM/mM ,’ 7.5 x 104 lCPM/mM // I / <7 / @1/ \LZ/ \) H3C(CEC)2-Z/ \) -c:ccrro + TCHO ,’ s s s s / ES 7.25 x 105 CPM/mM 1.94 x 104 CPM/mM o CPM/mM (/ \)—Z/ \B-CECCT(0H)CTH(OH) _ _ _[> (/ \u/ \B-CECCTO + TCHO s s s s 1.5 x 106 CPM/mM 5.9 x 105 CPM/mM 5.18 x 105 CPM/mM Biological material isolated by column chromatography and assayed. Chemical conversion produced the product which was assayed. 20 The pathway which is Shown in Chart 2 is consistent with the specific activities and demonstrates the existence of an enzyme system for the conversion of triple bonds to thio- phene rings, but it is not sufficient to distinguish between a preformed polyacetylene and another structure as the physiological precursor to terthienyl. The study of precursors to biological material can pro- vide evidence of the biosynthetic pathway, but a systematic degradation of the labeled biological material is required to ascertain the mode in which the precursor is incorporated into the biological product. The preliminary investigation of the degradation of terthienyl is one aSpect, therefore, of this investigation of polythienyls from Tagetes erecta. The reductive removal of sulfur is a reaction which has been known since 1940 (24). The first desulfurization of a polythienyl is the work of Wynberg and Logothetis (25). 5,5'-Diacetyl-2,2'-bithienyl and Raney nickel interact to form the simple structure, 2,11-dodecanediol. The diacetyl derivative of terthienyl is desulfurized with Raney nickel to form the correSponding long chain diol in 80% yield according to Wynberg (26). This is the only reported desul- furization of a terthienyl. The insolubility of the starting materials was particularly noted by these investigators. Numerous thiophene derivatives may be desulfurized in good yield (27,28). 21 The straight chain bifunctional structure obtained by desulfurization of 5,5"-—diacetylterthienyl is suitable for conversion to a dicarboxylic acid (26). Several pro— cedures for the degradation of fatty acids are available (29.50). The direct acetylation of terthienyl produces a mixture of the mono- and the diacetylated material. The maximum yields of the diacetylated and monoacetylated terthienyl are 51% and 40 to 50% respectively (26). The comparatively small amount of product which is obtained in this first step of the degradation sequence proposed above is undesirable. The direct introduction of a carboxyl function into terthienyl has not been reported in literature. Metal-hydrogen exchange does occur with thiophene and n-butyllithium (51). The addition of dimethylformamide to thienyllithium followed by hydrolysis yields the 2-thiophenecarboxaldehyde in 61%. Metal-hydrogen exchange with thiOphene and thiophene deriva- tives, followed by direct carbonation to give the carboxylic acid, or followed by the interaction with dimethylformamide to give the carboxaldehyde, are reactions which have been known for more than a decade (52,55). Equations 1 and 2 illustrate these reactions. 22 1 (55): 1gcog(s) Hooc-Z/ \B-CECCHg { \ 7 \ 2 HOH s / \ -CECCH3 c H Li Li-/ \ -CECCH3 57% D 1)BEF‘*\\1fl> s s ) 2 HOH OHC-Z/ \) -C_=.CCH3 s 21% 2 (51): Z/ \5 1) C4H9Li {> (/ \) -c00H s 2) C02 ( s) s 5)H0H 87% N z/ t... 2) DMF s 5)HOH 61% The metal-hydrogen exchange reaction with terthienyl and n-butyllithium desulfurization with Raney nickel, and the degradation of a long chain carboxylic acid constitute the preliminary study of a degradation sequence undertaken in the present study. 22 1 (55): 1)C02(s) HOOC-Z/ \B-CECCHg Z/ \5 1/ \5 2W 8, -CECCH c H Li Li- -CECCH 57a 3 4 9 9 31M 6 s ) 2 HOH OHC-z/ \) -CECCH3 S 21% 2 (51): Z/ \3 1) C4H9Li :> Z/ \) —CO0H S 2) C02 ( S) S 5)HOH 87% ,\. z/ t... 2) DMF s 5) HOH 61% The metal-hydrogen exchange reaction with terthienyl and n-butyllithium desulfurization with Raney nickel, and the degradation of a long chain carboxylic acid constitute the preliminary study of a degradation sequence undertaken in the present study. EXPERIMENTAL AND RESULT S Preparation of the Plants Seeds of the Tagetes erecta L. variety of marigold were obtained from W. A. Burpee Company, Fordhood Farms, Doylestown, Pennsylvania. Plants were grown from seeds in a greenhouse for six weeks, and then transferred to the laboratory. There, they were grown in nutrient solution with appropriate lighting, ambient temperature and humidity for 1 to 5 weeks. Growth conditions for all experiments are summarized in Table 9. The nutrient solution was prepared from stock solu- tions (1 N) of ammonium dihydrogen phOSphate, potassium nitrate, calcium nitrate tetrahydrate, and magnesium sulfate. For the preparation of 1 liter of nutrient solution, 1, 6, 4, and 2 ml, reSpectively, of the stock solutions were com- bined and diluted with 987 ml.water. Plant roots initially grown in sandy soil were generally 80% removed in transferring the plants to beakers or Erlen- meyer flasks for hydroponic growth. The daily addition of 5 to 10 ml.of water or nutrient solution (water and nutrient solution were added on alternate days) supplied the oxygen and nutrient requirements for the regeneration and growth of the plants' root systems. Tap water and continuous aeration enhanced the volume of root growth. The aerial portion of 25 24 Table 9. Growth Conditions Experiment Age of the Period in Nutrient Number Plants (wks) Solution (wks) , Season 1 8.8 2.8 Spring 2 8.8 2.8 Spring 5 6.0 0.1 Winter 4 6.0 0.1 Winter 5 7.0 2.0 Winter 6 10 1.0 Fall 7 10 1.0 Fall 8 9.0 2.5 Spring 9 9.0 2.5 Spring 10 9.0 2.5 Spring 11 9.0 2.5 Spring 12 8.0 0.1 Spring 15 10 5.0 Spring 14 10 5.0 Spring 15 5.6 2.2 Spring 16 11 4.0 Fall 17 11 4.0 Fall 18 11 4.0 Fall 21 10 4.0 Fall 22 10 4.0 Fall 25 10 4.0 Fall 24 9.5 5.0 Winter 25 9.5 5.0 Winter 26 10 5.4 Winter 27 10 5.4 Winter 28 10 5.4 Winter 29 9 5.0 Summer 50 9 5.0 Summer 51 11 5.0 Summer 52 11 5.0 Summer 55 11 5.0 Summer 25 the plants grew rapidly and appeared healthy. Some develop- ment of the bud region occurred when the plants reached the age of 10 weeks. Plants of this age were generally used for the hydroponic administration of the isotopically labeled compounds. Administration of Labeled Compounds A stock solution of the radioisotope labeled compound under investigation was prepared in distilled water. The carbon-14 and sulfur—55 labeled compounds listed in Table 10 were commercially available (see Appendix) and chroma- tographically pure. The plants used in a given experiment were removed from the nutrient solution, the roots were rinsed well in distilled water, and the plants were placed in clean beakers. The number of plants used in a given experiment was determined by the number of plants available, and the total quantity of roots of the plants. The amount of activity diSpensed (Table 10) was assumed to be sufficient to flood all the biological pathways available to the radioisotope form administered. The calculated amount of stock solution (2 to 5 microcuries per plant) was administered by Spreading the solution by pipet over the roots of the plants. After an absorption period of an hour, nutrient solution was poured into the beaker, and the plants were treated as described above until the roots were harvested. 26 Table 10. Radioisotope Forms Administered Experiment Total CPMx10'JSSpecific Activity Number Isotope Form Administered (DPM/um x 10'6) L Sodium sulfate-35$ 0.672 76.2 2 Sodium sulfate-35$ 0.672 76.2 5 DL-Glucose-UL-14C 1.99 195 4 DL-Glucose-UL-14C 1.99 195 5 DL-Ornithine-2-14C 51.5 4.28 6 DL-Glucose-UL-l4c 16.9 9.99 7 DL-Glucose-UL-14C 16.9 9.99 8 Sodium malonate-2-14c 55.5 5.62 9 Sodium malonate-2-14C 55.5 5.62 10 Sodium malonate-2-14C 55.5 5.62 11 Sodium malonate-2-14C 55.5 5.65 12 Sodium malonate-2-14C 44.4 5.62 15 DL-Methionine-2-14C 16.0 2.55 14 DL-Methionine-2-14C 16.0 2.55 15 DL-Methionine-2-14C 24.0 2.55 16 Sodium pyruvate-5-14C 21.0 14.4 17 Sodium pyruvate-5-14c 15.8 14.4 18 Sodium pyruvate-5-14C 14.7 14.4 21 DL-Cysteine-sss 178 4.65 22 DL-Cysteine-SSS 125 4.65 25 DL-Cysteine-sSS 125 4.65 24 DL-Serine-5-14C 56.5 11.6 25 DL-Serine-5—14C 50.5 11.6 26 DL-Cystine-1-14C 47.5 17.2 27 DL-Cystine-1-14C 52.5 17.2 28 DL-Cystine-1-14C 50.0 17.2 29 Sodium Pimelate-7-14C 28.5 4.44 50 Sodium Pimelate-7-14C 28.5 4.44 51 DL-Methionine-2-14C 65.4 9.26 52 DL-Methionine-2-14C 65.4 9.26 55 Maionic-2-14c Acid 66.7 8.88 27 The possibility of fungal growth over the long period allowed for root growth and development in nutrient solution was considered. Test and control solutions (experiments 19 and 20) using root fragments of equal length and sodium pyruvate-5-14C revealed a 2% difference in the radioactivity recovered from the nutrient solutions prepared with and without fungicide. An experiment with whole plants showed the relative uptake of sodium pyruvate-5-14C in the presence and absence of fungicide (Table 11). Experiments 51 and 52 tested the effect of an antibacterial agent on the uptake of activity diSpensed. Harvest of Roots and Extraction of Polythienyls The plants were allowed to metabolize the administered compound 0.1-10 days. The plants were then removed from the nutrient solution, and the roots were well rinsed, first with water and then with 95% ethanol. The nutrient solution and washings were assayed for activity. Paper chromatograms of a qualitative amount of the concentrated nutrient solu- tions examined gave no blue fluorescent areas under ultra- violet irradiation. Therefore, the polythienyls were not exuded by the plant roots in a detectable amount. The roots were air-dried one hour, weighed, and macerated with 40 ml. of 95% ethanol in a Waring blendor. The macerated roots were extracted with about 200 ml. of refluxing ethanol (95%) .ucmmmum mEHom oQOuOmHOHUMH Ham Umucmmmummu UCSOM >UH>Huom usouumm anew .m>onm confluommp mm Ummcmmmflp mmB pmumumflcfleom mufl>fluom 0:90 .OHumu unmme Hum m CH QUHHOHno Afisflcflvamcflsv IOCHEMIdvmflnlmcmawsuwfimomo pom mUHHoHLU Esflcflofluhmawumo mo musuxHE m mm3 ammumuomnflucm 0:93 .Aoomflocmum com .mcmmfioo HMUHEmno aflcnowflamov mofloflmcsm mHQmHHm>m NHHMHUHoEEoo m mH cmuamnmm no 2 >©.a O¢.m dm.m, 0 mm mm.fi hm.m dm.m m.H an N.Nd $.md >m.a w.d pd $.mm ¢.mm OH.N 0 ma COHuSHOm ucmfluusz uomnuxm Hocmnum ASIOH x Zmov AmIOH X Emmy NmIOH x EQQV Hmnfidz poom Emu0h22mpmum>flu0¢ pcwoumm oomumuwmcflfip¢ Qamflumuomflflucd omUHUflmcdm ucmEflummxm '1' I mxmums HMflumuomm pom Homasm mo wosum .HH magma 29 in a Soxhlet extractor for approximately 20 hours. The ethanolic extract was assayed for radioactivity. The uptake of the radioactivity dispensed is shown in Table 12. Paper chromatography was successfully used to monitor the presence of the fluorescent polythienyls in the concentrated extract. The solvent was removed under reduced pressure and the residue was extracted with petroleum ether (50-600) or alternately dissolved in 50 ml. of 2% methanolic KOH and saponified. Saponification was carried out by gentle reflux on a steam bath for 50 minutes. An investigation by paper chromatography of the residue which was not immediately soluble in petroleum ether showed the residue to contain about one-third (52.2%) of the total terthienyl isolated (experiment 5). For this reason and because Horn (57) had shown by n.m.r. that triglycerides complicate the purifi- cation of terthienyl, the immediate saponification of the residue from the root extract was preferred. The saponifi- cation mixture was diluted with 200 ml. water and extracted with approximately 150 ml. of hexane or petroleum ether (50-600). The extract was dried one hour over anhydrous magnesium sulfate, and the solvent was removed under reduced pressure. The residue was dissolved in 10 m1. of hexane, and the solution was chromatographed over a column of alumina (Alcoa, F-ZO). 5O 00.a 00.0 0.0 0.0 00 0a.0 00.0 0.0 0.0 mm 00.0 06.0 0.m 0.N H0 00.N 0>.¢ 0.N 0.a on >.Nm 00.N 0.N 0.H 0m 00.0 00.a 0.5 a.a mm 0.0m 00.0 o.d 0.0 mm 0.00 «0.0 H.o m.a mm 00.6 I 0.a >.d 00 00.0 I 0.0 >.H «N 0.00 00.a 0.a 0.a mm «.00 a0.m 0.0 0.0 mm am.m 00.a 0.0 H.0 am Nw.> 00.0 0.0 0.0 ma 00.0 0.HH 0.m 0.H ha 0¢.> 06.0 0.0 0.0 we 0a.0 00.0 0.0 0.0 0a 0a.0 00.0 0.H m.a dd 00.0 m.am 0.a 0.a mm 00.0 ma.0 0.0 0.0 0a «0.0 I 6.0 0.0 «a 0a.0 a0.a 0.0 9.6 0a 00.0 Ha.a 0.0 0.d 0 00.0 «0.0 0.0 0.0 0 m>.a am.0 0.0a 0.a m 00.0 I 0.0 0.a 0 I 00.0 0.0 0.0 0 I no.0 0.0 0.0 d I I 0.fi 0.0 0 I 0.00 0.0a 0.0 m I 0.00 0.d 0.0 a coausaom ucmfluusz uomnuxm Hocmnum Am>mpv Coaumm A00 muoom umnadz Eoum Umum>oowm Wufl>fluo¢ mo ucmuumm compansocH mo unmflwz unweflummxm >Ufl>fl¥UmOHUmm MO mxmums oNfi GHQMB 51 Purification Procedures The purification of biosynthetic terthienyl to constant specific activity was accomplished by one of three procedures: (a) repeated column chromatography of the crude biosynthetic terthienyl diluted with nonradioactive terthienyl, (b) the synthesis and isolation of the 5-acetyl derivative of the diluted sample of terthienyl, or (c) the fractionation of the marigold root extract over an alumina column followed by successive thin layer chromatograms of the column eluant fractions showing fluorescence under ultraviolet irradiation. The heterocyclic, 5-(5-buten-1-ynyl)-2,2'-bithienyl, was isolated for assay only by the latter method. Column packings of cellulose, silica gel, and activated alumina (Alcoa, F-ZO) were examined to determine the most efficient column packing. The columns were prepared in the usual manner. Methanol-water (60-40), hexane and hexane- diethyl ether (95-5), reSpectively, were the developing solvents employed. The eluant fractions containing terthienyl were identified by the isatin test (61). Columns of alumina, 10 and 50 g., were found superior, based on the fractionation of the root extract effected by adsorption on alumina, the time required, and the ease of recovery of terthienyl by removal of the solvent. The frequency distribution of activity in the eluant fractions of experiments 16 and 17 when one-fifth of the total root extract was chromatographed is shown in the chart on page 52. The eluant fractions 52 Chart 5. Frequency Distribution of Radioactivity Net CPM Net CPM 480 - General Comments: Experiment 16 460 ” Experiment 17 '-- Sodium pyruvate-5-14C 440 _ First column pass, crude extract (1/5) Fluorescent: 9-12 fractions‘(+ isatin) _ Support: alumina, 50 grams . 420 Solvent: hexane-ether Speed: 1 ml/min. 400 - Fraction: 10 ml. 580 - q .__I 560 _ 540 — 160 r 520 h I I 150 II 500 — I I 140 I | I I 280 — I I 150 I I II 260 T ' ' 120 I I 1 I I 240 r I I ,I 110 I I I I I I I 220 V I I l 100 I I I 200 _ : 1 __I 90 I : . 180 — I I I 80 : : . 160 —- I I : : : 7O , : : I I I 140 — I 'I—I I I 60 I I I I 120 r—: I I i I __, 50 I I I ' I I I I 100 -: ' ' l . I -- 40 I I L“ I ' I 80 :1 I I r1 : -_ 50 60 — I I I I " 20 L—1 .I r1 I I 40— LJ L“ L4 -~ 10 20 7‘, 0 10 12 14 16 18 20 22 Fraction Number 55 4 to 6 from a 10 g. column of alumina through which the solvent flow was 0.7 to 1 ml. per minute, and the volume/ fraction was 10 m1. showed a blue fluorescence under ultra- violet irradiation. The synthesis of the 5-acetyl derivative of terthienyl is reported in Table 15. The maximum recovery of 5-acetyl- 2,2';EV,2“’-terthienyl was 47.5%. The reaction mixture in carbon tetrachloride was chromatographed over a column of alumina. Carbon tetrachloride-diethyl ether was the eluting solvent. Thin layer chromatography using silica gel as the support was also applied. Table 15. Acetylation of Terthienyl Experi- Terthienyl Yield of Specific Activity ment Acetylated 5-Acetyl Terthienyl 5-Acetyl Number (micromole) Derivative Derivative (%) (CPM/0M) (CPM/0M) 2 46.8a 47.5 544000 520000 17 0.995a 28.0 422 (c) 18 6.65b 40.7 (d) (d) aReaction conditions: 800 for 18 hours in benzene with stannic chloride catalyst. bReaction conditions: 250 for 19 hours in benzene with stannic chloride catalyst. CThe expected activity calculated on the basis of yield was within the standard err0r of the backround. No activity was observed in the 5-acetyl derivative isolated from a thin layer chromatogram. Activity was observed in a 558 mu component. 54 Thin layers of silica gel H (Brinkman Company) or adsorbosil-2 (Applied Science Laboratories) were prepared on 8" x 8" glass plates. The silica gel support was activated by heating at 100-1100. The eluant fractions from an alumina column, which were fluorescent under ultraviolet irradiation, were concentrated to approximately 0.01 ml. A 5 to 5 drop quantity of purified benzene was added. The solutions were applied either by syringe or capillary bore pipet to a region about one-half inch from the base of the chromatoplate. The ideal fine line at the point of appli— cation was difficult to obtain. The thin layer chromatogram was developed with purified hexane in a sealed glass tank by ascending chromatography. The fluorescent areas on the thin layer chromatogram which were observed under an ultraviolet lamp were removed from the plate by an all-glass vacuum apparatus. The fluorescent materials, 5-(5Pbuten-1-ynyl)- 2,2'-bithienyl and terthienyl, were eluted from the silica gel with reagent grade diethyl ether. A solution of the polythienyl in 95% ethanol was prepared for assay, as was a blank. The recovery of terthienyl from thin layers of silica gel was limited by technique and not by significant retention or decomposition. A yellow coloration remained on the silica gel after elution of 5-(5-buten-1-ynyl)-2,2'-bithienyl with diethyl ether. Horn (57) observed the same phenomenon in his studies. 55 Table 14. Thin Layer Chromatography of Terthienyl Total micromoles Percent Total micromoles Percent plated ’ Recovered plated Recovered 0.0157 0 0.297 87.2 0.0296 55.2 2.58 95.4 0.0592 94.6 5.18 85.6 0.148 76.4 9.52 85.9 The polythienyls isolated from the roots of the mari- golds were identified by the observed chemical and physical properties (Table 15). A component absorbing at 552-554 mu was present in the polythienyl fractions in experiment 26 after the undiluted material was passed through a column of alumina once and chromatographed once on a thin layer of silica gel. The concentration of this component appeared greater in the bithienyl fraction. A contaminant absorbing at 556 mu was present in the polythienyl fractions of experiments 51 and 55 on the second thin layer chromatogram. No absorption at 556 mu was observed on the third chromatogram, nor was absorption in this region observed in experiments 24, 25, and 52. I Autoradiograms were made from the first thin layer chromatograms of the column fractionskfrom the D1 sample in experiments 21, 22, and 25, and of the column fractions of the undiluted sample in experiments 24 and 25. Fast medical x-ray film was laid directly on the surface of the 56 Table 15. Identification of Biosynthetic Polythienyls Observation Terthienyl 5-(5-Buten-1-ynyl)- Rf 2,2'-bithieyyl Ratio Isatin test violet to wine reda blue-green Permanganate test negative positive Rf (paper) 0.65 0.75 1.19b Rf (silica gel) 0.49 0.58 1.1711C d e Amax (mu) 550 544 aThis material was oxidized to 2,5-thiophene dicarboxylic acid (5,15). be's already reported give a ratio of 1.18 (5). 0This is an average of two observations. A maximum of 550 mu has been reported in ethanol and syn- thetic material has been observed to absorb at 552 mu in ethanol (2). eBohlman has reported a maximum of 545 mu in ether (46). chromatoplate. A glass plate held the film firmly against the silica gel surface for 92 hours. The x-ray film was developed by automatic processing. Figures 1, 2, and 5 are photographs of the autoradiograms of experiments 21, 22, and 25. The pertinent data are reported in Table 16. The posi- tive (+) marks on figures 1 and 5 were areas on which phos- phomolybdic acid was reduced (62). Terthienyl on silica gel did not affect this reagent; however, 5-(5-buten-1—ynyl)- 2,2'-bithienyl readily reduced a dilute solution of per- manganate. There was no reduction of the x-ray film 57 Mg. 1 11-12 9-10 Mg. 3 1-9 10—13 Coin-n Pncuonn 9-10 Calm Friction. Export-Int 23 (D1) is“; ‘ 1. 6—8 Colun Fractions Export-ant 21 (DI) 58 Table 16. Autoradiogram Data Experiment Area Wavelength Concentration Activity Number Number (mu) (micromoles) (CPM) 22 1 552 0.288 6920 2 540i4 0.0457 10000 5 556Sh - 2550 4 540 0.0750 15700 25 1 556i4sh - 2790 2 5528h - 2660 5 550 0.504 18900 4 545 0.164 52800 21 1 - - 1160 2 - - 1920 5 552 0.282 24600 4 544 0.260 76500 24 5 552 0.0670 60 4 545 0.0705 92 25 5 552 0.155 89 4 544 0.0555 51 emulsion in area number 5 on the autoradiograms of experi- ments 24 and 25 and only slight reduction in area 4. The phosPhomolybdic acid reagent (62) is sufficiently sensitive to detect 10-0.1 ug. of reductants, but these substances did not reduce the film in experiments 21 and 22. The radio- activity present in the polythienyl areas reduced the silver emulsion of the film: however, 5-(5-buten-1-ynyl)-2,2'- bithienyl also caused some chemical reduction of the film. Paper chromatography was employed to monitor the purity of terthienyl samples from repeated column chromatography. 59 A qualitative amount of sample was Spotted about 1" from the bottom of a Whatman #1 filter paper strip, 8" x 5", and developed by ascending chromatography in a cylindrical Specimen jar. Methanol-water, 60-40, was the solvent system. The bithienyl component (Rf 0.75) was distinct from terthienyl (Rf 0.65). Quantitative paper chromatography was examined using a tank, 24" x 12", fitted for ascending chromatography. The latter technique, however, was not suitable over a wide range of concentration. Measurement and Determination of Concentration Concentrations of terthienyl and 5-(5-buten-1-ynyl)-2,2'- bithienyl were obtained by the measurement of absorbance at 550 mu (e = 2.41 x 104) and 544 mu (e = 2.78 x 104)* reSpectively. The solvent used was 95% ethanol. Absorbancies were determined with a Beckman DB recording spectrophotometer. The concentration of 5-(5-buten-1-ynyl)-2,2'-bithienyl calculated from the absorbance at 544 mu was the concentration of the biosynthetic material directly isolated and purified by column and thin layer chromatography. The determination of biosynthetic terthienyl was accom- plished by isotope dilution analysis and by the direct observation of the material isolated by chromatography. * The molar absorbancy index at 541 mu calculated in hexane is 2.81 x 104 (57). 40 The double dilution method of Mayor and Collins (65) is an application of isotope dilution analysis to the determin- ation of yield and activity of radioactive compounds. A known amount (D1) of nonradioactive terthienyl was added to the ethanolic root extract after saponification, afid it was diluted tx> 10 ml. Half of this solution was removed and a second and larger quantity of nonradioactive terthienyl (D2) was added to one five-milliliter portion. Both portions, D1 and D2, were purified to constant Specific activity by procedures already described. From the relationships X -' A A "' 2 and A0 '- the amount of biosynthetic terthienyl isolated (x) and its specific activity (A0) were calculated. Successful appli— cation of this method was found to depend on three factors: (a) the selection of D; such that D; closely approximated x, (b) that D2 be five times greater than D1, and (c) that Al be larger than A2. Negative or unreasonably large values of x were obtained when these requirements were not met. Two sample experiments are reported in Table 17. The direct observation of the amount of purified bio- synthetic terthienyl was made possible by the selection of a satisfactory thin layer support and a solvent system for chromatographic separation. The accuracy of the calculated concentration of terthienyl was not limited by any 41 Table 17. Double Dilution Method Weighta Estimated Experi- of umoles ment roots of ter- Double Dilution Calculated Number (g .) thienyl D1(I1M) D2( 0M) ‘XfuMT‘ 16 2.9 1.02 0.96 4.80 0.905 17 1.6 0.56 0.96 4.80 -0.248 aThe roots were air-dried one hour before being weighed. This estimation was made by assuming 0.55 uM of terthienyl isolated per gram of root. estimations but did involve the direct measurement and ‘prbcessing of small concentrations. Nonetheless, this method provided the most reliable values of Specific activities. For experiments in which the double dilution method and the direct observation of concentration had not yet been implemented or were inapplicable, a third means of arriving at a concentration of biosynthetic terthienyl was employed. This was based on the observation that marigold roots, 0.5-1.5 g., which were rinsed in 95% ethanol and air-dried one hour at room temperature averaged 0.55 uM terthienyl per gram of root on one fractionation of the root extract over a column of alumina (Alcoa, F-20). The root extracts to which this method was applied were diluted with a large amount of nonradioactive terthienyl relative to the estimated 42 amount of biosynthetic material and purified by chroma— tography to constant Specific activity. Measurement of Radioactivity Radioactivity measurements (64,65) of purified terthienyl and 5-(5-buten-1-ynyl)-2,2'-bithienyl were made on thin layers of the compounds. Analytical solutions of the polythienyls were prepared in 95% ethanol and an aliquot of each was plated on an aluminum planchet. No correction of the counting data to infinite thinness was required for the samples of terthienyl (2,5). All samples prepared for counting were 5.05-0.02« uM/cma. It was assumed on the basis of sample size that no significant correction factor was required for thin layers of 5-(5-buten-1-ynyl)-2,2'-bithienyl of 0.05-0.02 micromole. If geometric efficiency for the radiation has been determined, errors of the order of 5% can be expected for essentially weightless sources and may be as large as 20% according to Snell (66). The uniformity of composition of the samples of terthienyl was shown by the stability of dilute solutions of terthienyl over a five-month period, of the solid pro- tected from direct light, and of the solution irradiated at 220 mu for 40 minutes and 550 mu for 5 hours. All planchets were counted in a windowless gas flow proportional counter. A Baird-Atomic model 155 scaler and argon -methane gas were used. The planchets were generally observed to a 2% probable error in the observed activity. 45 Decay losses for sulfur—55 samples were corrected. No correction for decay was required for carbon-14 samples. A secondary standard was used to monitor the daily per- formance of the counting arrangement. The observed efficiency for BaC03-14C was calculatedam524.9% (Tracerlab standard, 6070 dps). In addition to thin layer counting, purified samples of terthienyl were assyed as BaC03-14C for experiment 11. The biosynthetic material was diluted with two quantities of nonradioactive terthienyl according to the double dilu- tion method previously described. The samples were purified to constant Specific activity as assayed by thin layer counting. An aliquot of the ethanolic solution of each dilution sample (D1 and D2) was evaporated to dryness in a wide mouth standard tapered joint tube and oxidized accord- ing to the Van Slyke-Folch method (67). The liberated carbon dioxide wds precipitated as BaC03 from a saturated solution of Ba(0H)2, collected by vacuum filtration, and reprecipi- tated once. The BaCOs samples were dried at 1100, cooled in a desiccator (ascarite), mounted on aluminum planchets, and counted in a gas flow windowless proportional counter (Nuclear Chicago model 192). Activity was observed to 10,000 counts at the beta plateau voltage. The observed rates were corrected for self-absorption to zero sample thickness. The correction factor applied was obtained from an empirical calibration curve constructed for the Nuclear 44 Chicago instrument from the data of the fraction of maximum activity observed and the corresponding density for samples of BaCOs of known specific activity. Table 18. Experiment 11: Barium Carbonate Counting Diluted Samples Specific Activitya Specific Activityb Terthienyl BaC03 Terthienyl D1 4,700 5,750 D2 5,580 5,120 aCorrected to zero sample thickness, CPM/mM. bCalculated as CPM/mM by thin layer counting with no cor- rection for self-absorption. Dilution Factor The dilution factor has been used to express the efficiency of incorporation of an administered carbon-14 or sulfur-55 labeled compound into biosynthetic terthienyl and 5-(5—buten-1-ynyl)-2,2'-bithienyl. The observed activities of all samples of biosynthetic material were corrected for the efficiency of the counting arrangement to carbon-14 radiation. The efficiency of the counter to sulfur-55 radiation (Eavg 0.05 mev) was assumed to be the same as that observed for carbon-14 (Ea vg 0.05 mev). The dilution factor is defined as the quotient of the specific activity (DPM/uM) of the biosynthetic material and the Specific activity (DPM/uM) of the administered radioisotope form. The dilution factors determined are summarized in Tables 19 and 20. 45 Table 19. Incorporation Data for Terthienyl . Probable Experi- Specific Standard ment Activity Error cog Dilution Number Isotope Form CPMZuM S. A. Factor 1 Sulfate-356 161000: 118 2 Sulfate—358 544000a 55.1 5 Glucose-14c 1050 46600 4 Glucose-14c 5740? 12900 5 0rnithine-14C g - 6 Glucose-14c 141 17600 7 Glucose-14c 548a 4560 8 Malonate-14c 402a 2260 9 Malonate-l4c 76.9b 11800 10 Malonate-14c 112b 8060 11 Malonate-14c 79.4b 11500 12 Malonate-14C e - 15 Methionine-14C 557a 1040 14 Methionine-14C 580a 1520 15 Methionine-14C e - 16 Pyruvate—i 4C 174C 14.5 20600 17 Pyruvate-14C 74.5a 47600 18 Pyruvate-14C e - 21 Cysteine-35S 80400C 5700 14.4 22 Cysteine-35S 19000c 2020 60.8 25 Cysteine-35S 500000C 14800 5.86 24 Serine-14C 824d 20.2 5550 25 Serine—l4c 605d 88.5 4820 26 Cystine-14C e - 27 Cystine-14C 1500d 514 5500 28 Cystine-14C e - 29 Pimelate-14C e - 50 pimelate-14c 2490b 594 445 51 Methionine-14c 1670d 544 1585 52 Methionine-14C e - 55 Maionic-l4c acid 1185b 141 1865 aThe method employed the assumption of 0.55 0M terthienyl obtained per gram of root after one fractionation of the root extract over alumina. The concentration was ascertained by direct observation on two purification processes followed by dilution and further purification. CThe double dilution method was applied to obtain the concentration of biosynthetic terthienyl. The concentration was directly observed on purification to constant specific activity. eNo activity was observed on exhaustive purification. 46 Table 20. Incorporation Data for 5-(5-Buten-1—ynyl)-2,2'- bithienyl Probable Experi- Specific Standard ment Activity Error of Dilution Number Isotope Form CPM/0M S. A. Factor 21 Cystine-ass 517000 4590 5.66 22 Cystine-SSS 167000 4250 6.94 25 Cystine-ass 299000 4500 5.87 24 Serine-14C 1500 166 2220 25 Serine-14c 925 55.4 5140 26 Cystine-l4c 441 150 9780 27 Cystine-14C a - 28 Cystine-l4c b - 29 Pimelate-14C b - 50 Pimelate-14C 2180 486 509 51 Methionine-14C b - 52 Methionine-14C b - 55 Malonic-14C acid b - aThe concentration was too small to determine Spectrophoto— metrically. bNo activity was observed on exhaustive purification. 5-(2,2'-Bithenoyl)propionic Acid A solution of 2,2'-bithienyl (25), 5.00 g. (0.018 mole), in 20 ml. of thiophene-free benzene was mixed with 2.71 g. (0.020 mole) of freshly prepared B-carbomethoxypropionyl chloride (68) at room temperature in a 50 ml, round- bottomed flask equipped with a calcium chloride drying tube and magnetic stirring bar. The stirred mixture was cooled to 00 , and 2.1 ml. (0.018 mole) of reagent grade anhydrous stannic chloride in 10 ml. of benzene was added dropwise. The reaction mixture was agitated at room temperature for 47 one hour after the stannic chloride was added to complete the reaction. A dark green material was observed to collect on the walls of the flask and then dispersed in the reaction medium. The reaction mixture was cooled to 100 and hydrolyzed with dilute hydrochloric acid. The aqueous layer was separated from the organic layer, ex- 5racted with diethyl ether, and the aqueous layer was discarded. The combined ether extracts and benzene solu- tion were washed with water and 5% aqueous sodium bicarbonate and dried overnight over magnesium sulfate. The solvent was removed at reduced pressure on a rotary evaporator. The tan residue (4.5 g.) melted at 69-770. Column chromatography and recrystallization from methanol gave a light yellow solid which melted at 86-870 (25.5%). The infrared spectrum (KBr pellet, Beckman IR—5) confirmed the presence of an ester function by the carbonyl absorption at 1740 cm‘l, the ketone function by a band at 1650 cm'l, and a monosub- stituted bithienyl residue by the absorption bands at 840 cm‘1 and 805 cm-1. The molar absorbancy, 21000, and the absorption maximum, 549 mu (95% ethanol, Beckman DU), were consistent with a 5-acyl-2,2'-bithienyl. The ester was hydrolyzed with aqueous sodium hydroxide and the tan product was isolated by precipitation with hydrochloric acid and extraction with ether. Recrystal- lization from dioxane gave light yellow needles, m.p. 169-700. The infrared spectrum, Figure 4, (KBr pellet, 48 I I I I I I I I 5000 1720 1645 1450 1255 1075 840 792 cm'1 Fig. 4. Infrared Spectrum of 5-(2,2'-Bithenoyl)propionic Acid VF I I L I J I 5100 1650 1450 _1281 1081 856 805 cm'1 Fig. 5. Infrared Spectrum of 5-Acetyl-2,2'-bithienyl E . 11 I. l , 5050 1651 1459 1280 ,855 796 cm‘1 Fig. 6. Infrared Spectrum of 5—Acetyl—2,2';5',2"—terthienyl 49 Beckman IR-5) showed a broad absorption band at 5540-2500 cm-l, a carbonyl absorption band at 1720 cm'l, and the monosubstituted bithienyl residue absorption bands at 840 cm’1 and 792 cm-1. The ultraviolet spectrum displayed the expected 549 mu band and a band of lesser intensity at 245 mu (95% ethanol, Beckman DB). The nuclear magnetic resonance Spectrum (Varian A-60, dimethylsulfoxide-ds) exhibited a carboxylic acid proton signal at -2.08 tau and aromatic protons' multiplet centered at 2.45 £32_with the low field doublet at 2.04 tag, The calculated coupling constants for the aromatic protons compare to 0.05 c.p.s. with the coupling constants which were measured for 5-acetyl-2,2'-bithienyl (J34=4.10, J3-4'=5.75, J4'5'=5.10. J3'5-=1.20 c.p.s.). Anal,” Calcd. for C12H108203: C, 54.2; H, 5.76. Found: C, 54.5, H, 4.82. 5-Acetyl-2,2'-bithienyl 2,2'-Bithienyl, 5.00 g. (0.018 mole), and reagent grade acetyl chloride, 1.42 g. (0.018 mole), were mixed in 20 ml. of thiophene-free benzene at room temperature in a 50 ml. round-bottomed flask equipped with a magnetic stirring bar and calcium chloride drying tube. Stannic chloride, 2.1 ml. (0.018 mole), was added at 00 and the acylation performed in the manner previously described. Product iso- lation gave a solid, 2.75 g, m.p. 105-1080. The yellow solid failed to sublime at 1000/11mm. Column chromatography and recrystallization from 95% ethanol raised the melting 50 point to 114-1150 (literature value, 114-1150) (69). The ultraviolet spectrum (95% ethanol, Beckman DB) exhibited an absorption maximum at 550 mu (19800) and an absorption band of lesser intensity at 245 mu (70). The nuclear mag- netic resonance spectrum (Varian A-60, dimethylsulfoxide-ds) revealed a multiplet centered at 2.49 t23_with a low field doublet at 2.09 Egg, The coupling constants were calculated as J34=4.05, J3-4'=5.80, J4'5I=5.00, J3I51=1.25 c.p.s. The infrared Spectrum, Figure 5, was consistent with the known structure and was used to confirm the structure of 5-(2,2'-bithenoyl)propionic acid. 5-Acetyl-2y2'j5',2"~—terthienyl Terthienyl (1), 0.519 g. (1.2 mmoles), was dissolved in 100 ml. of thiophene-free benzene in a 250 ml. round- bottomed flask equipped with a calcium chloride drying tube. Reagent grade acetyl chloride, 0.5 ml., was added. Three drops (1.1 mmoles) of reagent grade anhydrous stannic chloride were added by pipet to the stirred reaction mixture. The reaction mixture was heated at its reflux temperature for 18 hours and then hydrolyzed with dilute hydrochloric acid. The benzene solution and ethereal extracts of the aqueous phase were washed with water and saturated sodium bicarbonate and dried in contact with anhydrous sodium sulfate overnight. The residue obtained on evaporation of the solvents was chromatographed over ten grams of alumina (Alcoa, F-20). Unreacted terthienyl, 82.1 mg. (25.6%), was eluted in 51 fractions 4—5 (10 ml.) with petroleum ether-diethyl ether. The second component eluted from the column with diethyl ether was rechromatographed. The material eluted with carbon tetrachloride-diethyl ether was sublimed. The yellow sublimate, 99.1 mg. (57.2%), melted sharply at 1720. The ultraviolet Spectrum revealed the expected absorption maximum at 592 mu (5) and the infrared spectrum (KBr pellet, Perkin Elmer Model 21) exhibited the diagnostic absorption bands at 1651 cm‘l, 855 cm-1, and 796 cm‘l. Attempts to implement the procedure of wynberg (25,26) were unsuccessful. Higher temperature acetylation with a Lewis acid was known to give a lower yield of monoacetylated polythienyl (69). Repetition of this procedure with 1.4 mmoles of terthienyl and two drops of stannic chloride yielded 51.0% of unreacted terthienyl and 40.1% 5-acetylterthienyl. 2,2'-Bithienyl-5-carboxylic Acid The experimental procedures of Taft (51) and Skatteboel (55) were followed. 2,2'-Bithienyl, 5.11 g. (18.7 mmoles), Was dissolved in 40 ml. of dry ether in a three-necked flask fitted with a reflux condenser, a magnesium perchlorate drying tube, a dropping funnel, a nitrogen inlet tube, and a mechanical stirrer. The solution was cooled to -700 by immersion in a dry ice-acetone bath, and n-butyllithium, 15.5 ml. (20 mmoles), in hexane was added dropwise during 10 minutes, The reaction solution was allowed to warm to room temperature by removing the dry ice-acetone cooling bath. . .- - 52 The temperature of the reaction mixture was increased to 26—500 and then cooled again to -700. Dimethylformamide, 5.0 ml. (59.0 mmoles), was added dropwise. The reaction mixture was Slowly brought to room temperature following the addition of the amide and set aside at room temperature for 18 hours. The lithium salt was hydrolyzed with a saturated :3 solution of ammonium chloride. A solid product was insoluble in either phase. The solid obtained from the ethereal solu- tion was oxidized with silver oxide (71). The crude acidic ‘2;l"__ material, m.p. 174-6O (56.7%), was recrystallized from methanol, m.p. 185-40 (72). The solid was soluble in ethanol and methanol and only slightly soluble in hexane. Desulfurization of 2,2'-Bithienyl-5-carboxylic Acid The carboxylic acid, 1.6 g. (7.6 mmoles), was dissolved in dimethylformamide, 100 ml., in a Parr pressure bottle, and 12 g. of W-2 Raney nickel was added.* The Parr vessel was filled with hydrogen, 52 p.s.i. (72.5 mmoles) at 250, and shaken while the reaction temperature was increased by external heating to 850. The decrease in hydrogen pressure after 20 hours was 1 p.s.i. (1.59 mmoles), and after 40 hours, it was 15 p.s.i. The reaction mixture was allowed to settle, and the solvent was decanted. The Raney nickel was washed with petroleum ether and filtered. The organic *Private communication from D. Anderson, Department of Chemistry, Michigan State University. 55 solutions were combined and poured into 250 ml. of water. The diSpersion was extracted with four portions of petroleum ether (200 ml.). There was a great deal of sludge remaining in the separatory funnel. The extract was dried over mag- nesium sulfate, the solvent was evaporated, and the residue was esterified with methanol-sulfuric acid. Distillation of the esterification product after the usual isolation gave a colorless distillate, 0.2 g., distilling at 65-900/1 mm. of mercury, which fluoresced blue under ultraviolet irradiation. A sodium fusion test showed sulfur to be present. Vapor phase chromatography of 0.1 ml. on a six-foot Apiezon L column at a column temperature of 1910, attenuation of four and helium flow rate of 2 ml. per minute showed the presence of two components having retention times of 6.0 and 28.8 minutes reSpectively. The relative peak areas were 1:5.8. The retention time of 6.0 minutes corresponded to that of an authentic sample of methyl pelargonate. The sample size was insufficient for collection and characteri- zation by infrared Spectra. 5,5" -Terthienyldicarboxylic Acid To a vigorously stirred solution of 4.04 mmoles of terthienyl in 200 ml. of dry ether at -550 was added 5.5 ml. (8.08 mmoles) of n-butyllithium in hexane. At temperatures below -55°, terthienyl precipitated. Tetrahydrofuran gave better solubility. The dropwise addition of the organo- lithium reagent required 5 minutes. The yellow reaction 54 solution became cloudy, and a yellow precipitate formed in 6 to 10 minutes. The dry ice-acetone bath was removed, and the reaction mixture was stirred at room temperature until it reacted (50 minutes). The reaction mixture was again cooled to -400 and dimethylformamide, 1.0 ml. (15.0 mmoles), was added dropwise. An instantaneous deepening .m of the color of the precipitate to an orange hue occurred. The reaction mixture was stirred overnight at room tempera- ture after the addition of the amide to complete the re- action. Hydrolysis of the lithium salt was effected by the addition of 50 ml. of saturated ammonium chloride solu- tion. The ether was removed from the reaction vessel on a rotary evaporator, and the precipitate was separated from the aqueous phase by vacuum filtration. A positive dinitrophenylhydrazine test was obtained with the red-brown solid. The solid, 1.55 g., was added in portions to a cooled slurry of silver oxide (1.56 9. silver nitrate and 0.54 g. of sodium hydroxide in 10 ml. of water), and the reaction mixture was stirred for 10 minutes. Since particles of the red-brown solid were still visible, 25 ml. of tetra- hydrofuran were added. Vigorous stirring of the reaction mixture was continued for 20 minutes. A silver mirror was observed on the walls of the flask. The reaction mixture was filtered, and the residue from the oxidation reaction was extracted overnight with tetrahydrofuran. The solvent was removed under reduced pressure, and the solid was 55 washed with dilute acid. The filtrate from the oxidation reaction was acidified with dilute acid, the tetrahydrofuran was removed on the rotary evaporator under reduced pressure, and the solid was collected and washed with dilute acid. The amount of total solids recovered was 0.9071 g. The solids were slightly soluble in ether and quite soluble in benzene and glacial acetic acid. Purification on columns of silica gel gave three fractions. The fractions eluted with hexane, hexane-ethyl acetate, and glacial acetic acid were yellow, orange, and red respectively. The yields in relative percent by weight were 15.4, 79.7, and 4.87 respectively. The yellow fraction, m.p. 79-820, was identi- fied as terthienyl by the Rf (0.588) on chromatostrips and ultraviolet absorption maxima after purification on thin layer preparative chromatograms. The contaminant present in this fraction, a yellow fluorescent substance (Rf 0.12), traveled much slower than terthienyl when hexane was used as the mobile phase. The orange fraction was a mixture of four components in addition to terthienyl. The latter was present in 4.6% by weight. The four components separated by thin layer chromatography on silica gel with hexane to produce yellow fluorescent bands at Rf values 0, 0.068, and 0.125 and an orange band at 0.171. An infrared Spectrum (KBr pellet, Unicam SP .200) of the red fraction after sub- limation showed no absorption in the carbonyl region and bands at 852 cm‘1 and 811 cm‘l. The absorption band at 1650 cm"1 has no analogy in the Spectrum of terthienyl (75) and, 56 therefore, may be a carbon-oxygen stretching frequency in a highly conjugated system. The melting point, 101-1050, distinguished the material from a pure, higher polythienyl (74). Thin layer chromatography (hexane:ether:acetic acid ratio of 90:10:1) showed two major fluorescent areas, red (Rf 0.55) and yellow (Rf 0.51). The fiery red material gave an intense yellow-green fluorescence in 95% ethanol, and absorption maxima were observed (Beckman DB) at 404 mu, 260 mu, and 240 mu. Aqueous sodium hydroxide extracted a material from the orange fraction of the total solids which, after acidification of the aqueous solution, was easily soluble in benzene. The material was identical in behavior on chromatostrips and melting point (187-1900) to the com- ponent of the thin layer preparative chromatogram which did not leave the origin (sublimed at a bath temperature of 1900 and a pressure of 0.5 mm Hg.). Absorption maxima in 95% ethanol were observed at 568 mu and 260 mu (Beckman DB). The infrared spectrum of the yellow sublimate (KBr pellet, Unicam SP .200) showed absorption bands at 5500-2500 cm‘l, 1685 cm‘l, and 1520-1210 cm-l, and the characteristic 2,5- disubstituted thiophene bands at 855 cm‘1 and 811 cm‘l. The quantity of yellow material isolated from the orange fraction was 25.6% by weight. Thin layer chromatostrips developed in hexane:ether:acetic acid (90:10:1) exhibited a blue (Rf 0.15) and a yellow (Rf 0.51) fluorescent areas, notwithstanding the application of sublimation and chroma— tography. 57 m I Ilzggg I I II 5400 5070 1650 1465 852 811 cm‘l Fig. 7. Infrared Spectrum of the 404 mu Product 5070 III II I 11 I 5450 2950 1685 1660 1470 855 811 700 cm‘1 Fig. 8. Infrared Spectrum of the 568 mu Product 58 Desulfurization of 5,5" -Terthienyldicarboxylic Acid The crude orange solid, 66 mg., obtained from the silver oxide oxidation of the formylated terthienyl was dissolved in 200 ml. of dioxane. Raney nickel prepared from 50 g. of alloy was added (75). The reaction mixture was heated at its reflux temperature. Qualitative thin layer chromatography applied after 18 hours reaction (hexane:diethyl etherzacetic acid in 90:10:1 ratio in silica gel) showed no fluorescent materials under ultraviolet irradiation of the chromatogram. The Raney nickel was separated from the solution by gravitational filtration. The filtrate was concentrated to 100 ml. on a rotary evaporator and 100 ml. of water was added. The slightly basic solution was acidified with hydrochloric acid and extracted with ether. The ethereal extract was washed with water, dried over magnesium sulfate, and evaporated under reduced pressure. The residual oil was esterified with 25 ml. of methanol and 5 ml. of sulfuric acid in the usual manner. The esterification reaction products were distilled at reduced pressure over a short distillation path. The distillate, 0.15 ml., boiled at 150-1550/0.7 mm. (5400/760 mm). The infrared Spectrum of the neat sample was simple (Beckman IR-5, Figure 9). Absorption bands at 1740 cm‘l, 1200 cm'l, and 1170 cm’1 were assigned to the ester carbonyl stretching frequency and methyl ester carbon-oxygen stretch- ing frequency. Vapor phase chromatography on a six-foot 59 L I I L1 2940 1740 1448 1200 1170 812 cm‘1 Fig. 9. Infrared Spectrum of Dimethyl Tetradecanedioate II I II I I I I I 5075 1600 1450 1075 1050 755 755 700 cm-1 2985 1495 ‘* Fig. 10. Infrared Spectrum of 1,1-Diphenyldodec—1-ene (neat) 60 Apiezon L column at a column temperature of 2250 and helium flow rate of 2 ml. per minute showed three major fractions present in the distillate. The retention times of 6.6 minutes, 22.5 minutes, and 27 minutes correspond to relative peak areas of 2.94, 1.08, and 1.00 respectively. Attempted sample collection was unsuccessful because of the small initial sample size. The b.p. of the dimethyl ester of tetradecanedioic acid has been reported as 1960/9.5 mm. (5500/760 mm.) and 2020/14 mm. (5420/760 mm.) (76). Raney nickel prepared by digestion (77) for 5 hours in concentrated aqueous base was found to be ineffective in the desulfurization of a similar crude sample of terthienyl- carboxylic acid. Barbier—Wieland Degradation Sequence (78) The esterification of long chain fatty acids was accomplished satisfactorily by two procedures (79a,80). Table 21. Esterification of Fatty Acids — t Acid Quantity Conditions Reagents Yield (9.) - (%) Lauric 5.09 1008,1 hour CH30H,H2804 90.7 Hendecanoic 1.19 65 ,5 minutes CH30H,BF3 70.4 Hendecanoic 2.00 648,5 minutes CH30H,BF3 87.2 Hendecanoic 5.00 64 ,10 minutes CH30H,BF3 90.0 Hendecanoic 1.00 640,10 minutes CH3,OH,BF3 85.8 61 The CH30H-BF3 reagent was prepared by mixing 580 ml. of purified methanol, 0.6 ml. distilled water, and 57 g. of BF3 at 0-100. The reagent and carboxylic acid were mixed at room temperature in weight:volume ratios of 1:20, 2:40, and 5:60. The mixtures were heated on a steam bath for the period of time indicated in Table 21. Petroleum ether was added to the cooled reaction mixture; the diluted mixture was poured into a seven-fold excess of water, and the organic phase was separated. Petroleum ether extracts of the aqueous phase, 150 ml., were added to the organic phase separated above, washed with water, and dried over magnesium sulfate. The solvent was removed at reduced pressure, and the residue was distilled. Methyl laurate distilled at 1450/18 mm. over a Short distillation path. Methyl n-hendecanoate distilled at 810/1 mm. and 970/4.5 mm. over a short distillation path. Boiling points agreed with literature data (81). The ester carbonyl absorption band appeared at 1750 cm’1 in the infrared spectra (neat, Beckman IR-5). The Grignard reagent (79b), phenylmagnesium bromide (0.082 mole), was prepared observing the usual precautions. Methyl n-hendecanoate, 0.90-2.90 g. (0.0045—0.014 mole), in 10 ml. of dry ether was added dropwise to the vigorously stirred Grignard reagent at room temperature. A precipitate was observed. The reaction mixture was set aside at room temperature for about 18 hours. Product isolation gave 62 9.2—8.5% of biphenyl (removed by vacuum distillation at 980/2.8 mm.) and the crude tertiary alcohol. The alcohol, 100 ml. glacial acetic acid, and 50 ml. of redistilled acetic anhydride were refluxed for an hour. With the dis- tillation head adjusted for delivery, the acetic acid- acetic anhydride solvent was distilled (78-1200) until a volume of 50-60 ml. remained in the reaction flask. The crude olefin and solvent were cooled to 500. The distill- ing head was replaced with a parallel side arm connector fitted with a thermometer and dropping funnel. Chromium trioxide, 2-6 9. (0.020-0.060 mole), in 1.5-6 ml. water and 10-40 ml. glacial acetic acid were added in portions to the stirred solution at a rate sufficient to maintain the reaction temperature in the range of 50-600. After adding the oxidizing agent, the reaction mixture was kept at 500 for an hour and then at room temperature for 18 hours. Excess chromic oxide was destroyed with methanol. The side arm connector was replaced with a Claisen head, and the solvent was removed under reduced pressure until a paste remained in the reaction flask. Water was added, and the mixture was extracted with 500 ml. of diethyl ether in small portions. The ethereal extracts were washed with 10% aqueous sodium hydroxide until the washings were basic to hydrion paper. The combined ethereal extracts were then washed with water and dried over magnesium sulfate. Evaporation of the ethereal solution gave an immiscible 65 mixture of yellow and colorless oils. The mixture was chromatographed over 20 g. of alumina and column fractions 4 to 7 which were eluted with hexane and 5% ether in hexane gave the crude ketone, benzophenone (m.p. 59-400, 26%: literature m.p. 45—480) (82b). The yellow oil from the column which would not crystallize was treated again with chromium trioxide in glacial acetic acid. Product isolation from the neutral organic fraction and one recrystallization from petroleum ether increased the overall yield to 52.2% benzophenone (m.p. 44-460). Mixed melting point determin- ation with an authentic sample showed no depression. Basic extracts of the ethereal solution were acidified to a pH of 1 with concentrated hydrochloric acid, extracted with 500 ml. of ether in small portions, and dried over magnesium sulfate. The yellow oil obtained on evaporation of the ether solvent was esterified with methanol-boron trifluoride. The ester, methyl caprate (b.p. 60-660/1 mm.), was obtained (55.8% overall yield). The boiling point reported in literature was 2240/760 mm. (82a). The infrared Spectrum of the neat sample confirmed the ester structure of the distillate. The vapor phase chromatography retention time, 9.9 minutes, of the ester on an Apiezon L column at a column temperature of 1880 and helium flow rate of 2 ml. per minute correSponded to an authentic sample of methyl caprate. 64 Permanganate Oxidation of 1,1-Diphenyldodec-1-ene (85) 1,1-Diphenyldodec-1-ene (Figure 10) was obtained by the dehydration of the tertiary alcohol from the hydrolysis of the appropriate Grignard product. To the olefin in 50 m1. glacial acetic acid was added an excess of solid potassium permanganate in one portion, and the reaction mix- ture was heated to 750for a half hour. The reaction mixture was cooled to room temperature and poured into dilute sul- furic acid. The acidic mixture was heated on a steam bath, and the coagulated manganese dioxide was separated by vacuum filtration. The filtrate was cooled and the orange oil was taken up in ether. Extraction of the ethereal solution with aqueous sodium bicarbonate gave a trace amount (not measured) of solid. Product isolation from the ethereal solution gave a colorless solid (m.p. 85-870, 20%) which analyzed for the 1,2-diol. .5231: Calcd. for C24H3402: C, 81.20; H, 9.60. Found: C, 80.87; H, 9.55. The infrared Spectrum (KBr pellet, Beckman IR-5) exhibited a broad band at 5571-5555 cm‘l; the tertiary and secondary carbon-hydroxyl stretching frequencies, at 1176 cm‘1 and 1095 cm‘1 and the mono-substituted benzene ring carbon- hydrogen bending absorptions, at 750 cm'1 and 697 cm‘l. No further study of the products was undertaken. Attempted Beckmann Monooxime Degradation Sequence (84) Tridecanoic acid (Eastman Organic Chemicals white label) and a seven-fold excess of thionyl chloride (purified) were 65 mixed in a round-bottomed flask equipped with a magnetic stirrer and calcium chloride drying tube. The vigorously stirred reaction mixture was heated to 400 for a half hour. Excess thionyl chloride was removed under reduced pressure at room temperature. The yellow-tinted acid chloride was dissolved in a ten-fold excess of dry thiophene-free benzene and cooled to 00, and a slight excess of reagent grade aluminum chloride was added in portions. The reaction mix- ture was stirred at room temperature for 18 hours. Dilute hydrochloric acid was added. The organic layer was separated from the aqueous layer. Unreacted carboxylic acid was removed by extraction of the benzene solution with 1N sodium hydroxide. The organic layer was shaken with a 1:2 methanol-water solution to remove traces of the sodium salt of the carboxylic acid. After removal of the solvent from the benzene solution, the white crystalline ketone was recrystallized from hexane (m.p. 59-40.50). The melting point and infrared Spectrum agreed with literature data for the ketone (85). In four experiments using 0.546-2.00 g. of the carboxylic acid, 55-66% yields of the purified ketone were obtained. The ketone, 2.87 mmoles, was dis- solved in 10 ml. of purified dioxane and 0.6 ml. concentrated hydrochloric acid (diethyl ether and hydrogen chloride were used with one sample of ketone). The reaction mixture was heated to 500 (540 in the case of diethyl ether). A solu- tion of 0.49 ml. (5.9 mmoles) of freshly distilled iso- amylnitrite (purity by v.p.c. analysis, 76%) in 5 ml. of 66 purified dioxane was added dropwise during 45 minutes. The reaction solution was light brown in color and clear. After an additional 15 minutes heating at 500, 12 ml. (56 mmoles) of 5N sodium hydroxide were added. (Powdered sodium hydroxide was used with one sample of ketone.) The light brown organic layer separated from the aqueous phase. After cooling to room temperature, 2.0 g. (10 mmoles) of distilled p-toluenesulfonyl chloride were added in portions to the vigorously stirred diSpersion during 20 minutes. The diSpersion was warmed again to 500 and stirred for an additional 2 hours. The reaction mixture was cooled and poured into 500 ml. of water in a separatory funnel. The organic layer was separated, washed with water, and dried over magnesium sulfate. Lauryl nitrile was not detectable by boiling point range or infrared Spectrum (86) in the distillate obtained by reduced pressure distillation. ”The infrared spectrum had no absorption band between 5550-5000 cm‘1 and a well defined band at 1750 cm‘l. p-Toluenesulfonyl chloride was the only solid isolated from the organic phase. The aqueous phase was acidified with concentrated hydro- chloric acid and extracted with ether. The extracts were washed with water and dried over magnesium sulfate. The solvent was removed, and the residue was sublimed. Benzoic acid, m.p.120-1220, was obtained in yields of 9-19% in ‘four attempted degradations. DISCUSSION Biogenetic Speculations have the valuable function of providing a focus for the examination of possible path- ways for the biosynthesis of naturally occurring compounds. From principles of comparative structural analysis, economy, enzyme catalysis, and modern reaction theory, biogenetic pathways may be formulated. It is convenient to discuss the steps of a pathway in a certain order. The sequence of synthetic steps in the formulation of a natural molecule is impossible to discern from biogenetic considerations alone. Similarly, this inferential approach does not admit the identification of the actual reagents used but only the effective structural element involved. A satisfactory rationalization of the biogenesis of many plant products can be made by assuming an acetate-malonate chain followed by appropriate decarboxylation and condensation. The details of metabolism intermediary between the acetate-malonate primers and the derived natural products require the experi- mental evidence of radioactive tracer and enzyme studies. The biogenetic formulation of thiophenic natural' products from polyacetylenes conforms to the four criteria for a valid biogenetic hypothesis: comparative structure, economy, enzyme control, and conformity to modern reaction theory. 67 68 The functional group pattern and size of the poly- acetylenes which have been obtained from plants among the Compositae are comparable to the thiophenic compounds found in the same Family. The removal of a terminal methyl group which is formally required in some instances can be expected to proceed by oxidative elimination. Cell free extracts from the fungus, Coprinus quadrifidus, cause the elimination of the terminal functional group carbon of a ten carbon polyacetylenic alcohol to form a nine carbon polyacetylenic alcohol (10). HOOCCECCECC ECCH=CHCH2 OH ——DHC ECC ECC ECCH=CHCH2 OH The polyyne XI and compounds a.(11,59) and b (11) are clearly related. Compounds XII, c (45), and d (44) of Chart 4 diSplay a structural similarity. Chart 4. Naturally Occurring Polyynes and Related Thio- phenic Compounds XI . H3CCH=CHCECCECCECCECCH=CH2 a . H3CCH=CHCEC-Z/ \B -CECCH=CH2 S b. H2C(OH)3CH=CHCEC-Z/ \)-CECCH=CH2 S XI I . H3CCECC ECCECCH=CHCOOCH3 C . ch-Z/ \x -CECCH=CHCOOCH3 S d . Z/ \> -CECCH=CHCOOCH3 S 69 The oxygen functions in the compounds XIII and XIV from Artemisia arborescens (60) support Sorenson's (14) sug- gestion that polyketones are involved in the biosynthesis of the thiophene ring. Some oxidized sites are known, however, H0 H3CO H acco-Z/ \) -CECCH3 H3CCO- Z/ \) -CECCH3 5:, s S . I XIII XIV I to arise by secondary processes. Nemotinic acid (16) is labeled at alternate carbon atoms in biosynthesis of the 4) acid from acetate-1-14C. The 4-hydroxy substituent of the undeca- 5,6-diene-8A£Ldiynoic acid appears on an unlabeled carbon atom. The genesis of this atom is the methyl group of acetate-1-14C. Thiophenic compounds are presumably of secondary, if any, metabolic importance to the plants which produce them. The amount of material isolable is generally less than 0.02% by weight of the tissue extracted (Table 8, and references 57, 45, 46). The nematocidal effect of the poly- thienyl exudates of marigold roots has been observed (75,87), but‘the function of terthienyl in the blooms of the marigold, Tagetes erecta L., is unknown (1). It is reasonable, there- fore, to suppose that the plants use the fewest possible reactions to produce these materials. Economy would also suggest that the enzymes involved in the production of minor products be of a low order of Specificity. Enzymes merely 70 catalyze reactions that are sound mechanistically and are usually known to go 13 11339. The predominance of Egagg-addition to an acetylenic bond is mechanistically sound and experimentally demonstrable. Thiolacetone and methyl propiolate in the presence of an equimolar amount of base cyclize to 2-acetyl-5-hydroxythio- phene (88). The addition of thiolacetic acid to 1-hexyne at 00 with no initiation produces a total product yield of 55% which consists of the gig and traps products in 82:18 ratio. A» gig product is stereoselectively suitable for intramolecular interaction with an electrophilic triple bond for the formation of a five-membered sulfur heterocycle. It has been demonstrated in laboratory syntheses (19) that one thiophene ring may be formed by the addition of the elements of hydrogen sulfide to a triple bond; however, triple bonds adjacent to a thienyl function are unreactive under these laboratory conditions. Bohlman's work (4,5) unequivocally demonstrates the ability of a biological system to effect the conversion of acetylenic bonds to a vinyl thioether function and a thiophene ring. The formation of XV from XVI is shown CBHSCOCECCH=CHSCH3 CGHSCOCECCECH XV XVI to occur in Chrysanthemum segetum by the incorporation of the radioactive oxo-carbon atom of XVI into XV £1 vivo. 71 The incorporation of the tritium label of XVII,1,29H into XVIII, XIX, and XX in Echinops sphaerocephalus L. (Table 22) illustrates the biosynthesis of these thiophenic compounds from the pentyne. H3C/\/\ -CECCT=CTH -CECCTHCTH0C0CH3 S S S XXI XXII The tritium labeled pentyne XVII may (a) be diluted by the physiological precursor to XVIII, XIX, and XX; (b) be diluted by the pool size of the thiophenic compounds which is not likely; or (c) by the process of discrimination, be transformed only in part to XVIII, XIX, and XX with poly- meric decomposition and unknown metabolic routes accounting for the remainder of the twenty—five milligram initial dose. *- A precursor is any compound whether exogenous or endo— genous that can be converted by an organism into some product. An intermediate is a compound that is both formed and further converted by the organism under identical con- ditions (90). 75 The sequence of synthetic steps in the elaboration of a natural thiophenic compound will involve chain elonga- tion of some structural unit presumably by malonyl CoA, desaturation to acetylenic bonds, and the incorporation of a sulfur atom in ring closure to a thienyl moiety. Bohlman g5) has proposed biosynthetic Scheme A, Chart 5, to explain the degree of incorporation of the tritium label reported in Table 22. Bohlman's scheme assumes the requirement of a preformed polyacetylene of thirteen carbon atoms. Whether or not it is possible that chain elongation and/or desaturation occurs after the formation of one thio- phene ring is a mute question. Naturally occurring Chart 5. Biosynthetic Scheme A H3C(CEC)CT=CTH $ 9 H3C(CEC)2-Z/ \X-CECCTCICTHOCOCHS s / \ / \ -CECCT=CTH s / \ / \ -CECCTRCTHR' / \ / \ / \ S S S S S R = H R' = 0C0CH3 compounds XXIII, XXIV, and XXV which occur in Matricaria inodora (35) are suggestive of this latter mode of synthesis. 74 XXIII Z/ \B-CECCH=CHCH=CHCH=CH2 S XXIV [/ \>-CECCH=CHCH=CHCOOCH2CH3 S XXV ZZ—§>-CECCH=CHCH2CH2C00CH2CH3 S Compounds XXVI and XXVII from Ambrosia (25) and XXVIII and XXIX from Bidens (39) suggest that the formation of the thiophene ring occur across any suitably located triple bonds with preference for the center of a long carbon atom chain. This is consistent with the expectation that the interior triple bonds of a polyacetylene are more electrophilic than perpherial bonds with electronedonating groups and with the desaturation of long chain fatty acids which occurs about the ninth carbon atom (Confer Chart 1). The formation of H3CCEC-R"_l/ \S-CECR' I I S / a . H30(CEC) 2-Z/ \) -CECCTC1CTH0COCH3 S [H3c(c2c) 2-[_\> -cscc2c1] E{2C=C=CHCEC- / /\ -CECCT=CT% S S i i Fi(CEC)2-/ \ / \] a. H3C-/ \ / \—CECCT=CTH S S a,b. / \ / \ / \ a,b. Z] ))—Z/ \>-CECCT=CTH S S S S U) Q_—. a,b. / \ / \ -CECCTHCTHOH S a . Natural product from Echinops sphaerocephalus L. Natural product from Tagetes erecta and/or minuta. 92 The groups R and R' are introduced into Scheme B because the order of the steps in the sequence leading to acetylenic bonds is not confirmed. Rates k1 and k2 are assumed to be comparable although as long as both polythienyl pools are saturated with radioactivity from the hydrocarbon skeleton within 12 hours (Bohlman's experiment with tritium labeling in reference 5), this is not essential. Rate k3 is very different from k; and k2. There is no direct evidence of the-biological mechanism for the formation of a triple bond. Since acetylenic bonds adjacent to a thiophene ring are apparently stable towards addition 0f H28,xrelatiVe to other triple bonds (Table 5), it is not unreasonable to postulate desaturation to a terminal conjugated acetylenic system and the isomerization to the less stable allenic form before incorporation of sulfur and cyclization to a thiophene ring. Independent pathways for the polythienyls allow routes desig- nated by rates k; and kg to be about equally saturated with the first pulse of tritium, carbon-14 and sulfur-55 radio— activity.. The incorporation of further sulfur—55 along an independent pathway and the catabolism of terthienyl will account for the dilution factor of tritium labeled as well as sulfur-55 labeled terthienyl (3H:4140; 95$:60.8) compared to the bithienyl derivative (3Hz2000; 35S:6.94). The biological conversion, if any, of carbon-14 labeled 5-(2,2'—bithenoyl)propionic acid to naturally occurring thiOphenic compounds in Tagetes would contribute to the 95 clarification of desaturation, chain elongation, and cycli- zation on sulfur which can be effected enzymically. The synthesis of this bithienyl derivative has been reported for the first time in this work. The investigation of precursors to terthienyl and 5-(5-buten-l-ynyl)-2,2'-bithienyl in Tagetes erecta L. is SéJ! limited to the identification of compounds efficiently incorporated into the biosynthetic pathway. Elucidation of the mode of incorporation requires systematic degradation of the radioactive biosynthetic material and identification E? "‘ of the distribution of the radioisotope in the molecule. The relative stability of terthienyl with respect to the other naturally occurring thiophenic compounds is the determinant for its selection in a degradative investigation. The stability allows purification to constant specific activ- ity with minimal loss of terthienyl radioactivity and con- tamination by decomposition products. Any study of a reaction sequence to degrade terthienyl is necessarily concerned with‘ the conversion of the symmetrical molecule to a form which may undergo further and controlled degradation, and secondly, with the selection.of the route for the further degradation. The control of mono- and disubstitution in terthienyl can be accomplished reasonably well by control of reagent quantities and dilution. The acetylation procedure reported here has produced a 57-40% yield of the monoacetylated product with recovery of 25-51% terthienyl. This enables a yield of 94 at least 50% to be realized by recycling the recovered terthienyl. The maximum yield of diacetylated terthienyl reported is 51% (26). The chemistry of bithienyl which has been studied has established that substitution occurs in the 5,5'-positions when these positions are not substituted. The unique pattern in the aromatic region of the nuclear magnetic resonance spectrum, namely, the low field doublet and the sequence of apparent pairs of signals, provides a rapid and easily discerned characterization of the mono- and disubstituted polythienyl. The coupling constants are those reported for thiophene itself (58). The chemistry of terthienyl and higher polythienyls, on the other hand, has not been extensively studied. Direct metalation of terthienyl has precedence with the homologs, thiophene (51) and bithienyl (26). Two synthetic routes to the carboxylic acid are available from the thienyl- lithium. Carbonation with dry ice has been known to produce thiophenecarboxylic acids directly and in better yields (51, 55) than the alternate procedure of formylation followed by oxidation. 2,2'-Bithienyl-5-carboxylic acid synthesized by the latter route in this work is obtained in 52.7% in contrast to the 74% yield reported by the direct carbonation (26). Formylation with dimethylformamide.carbonyl-14C followed by oxidation, however, is a practical route to the carboxyl-14C acid, a derivative which is useful in the evalu- ation of the efficiency of subsequent degradative procedures. 95 The direct metalation of terthienyl with n-butyllithium, formylation of the polythienyllithium with dimethylformamide, and the silver oxide oxidation of the crude reaction product have been found to give a mixture of products. The difficult isolation of a pure sample of the carboxylic acid causes this reaction to be an impractical step in the synthesis of a h; terthienyl derivative suitable for systematic degradation. 3 This reaction and the direct carbonation of terthienyllithium may be a profitable study. The reaction mechanism (58) and Wynberg's metalation study on 2,5'-bithienyl (26) suggest the k: basicity of sulfur to be a factor in the reaction. The desulfurization of 5,5" -diacetylterthienyl and many other thiophene compounds have been reported in litera- ture (26). The activity of Raney nickel which is effective for a given thiophenic compound apparently varies. W-7 Raney nickel refluxed with 2,5-diphenylthiophene for six hours is reported to yield 45.9% of diphenylbutane and two other products (101). Raney nickel prepared rapidly at 00C, slowly brought to room temperature, and washed to remove the concentrated base is effective in the desulfurization of a crude sample of terthienyldicarboxylic acid. The infrared spectrum of the esterified product confirms the degradation to a long chain fatty acid. Two procedures for the degradation of fatty acids have been examined. Four criteria define an optimal procedure. It is desired (a) to degrade one carbon at a time, (b) to 96 obtain a good yield on a small scale, (c) to obtain the product in a form which is easily further degraded, and (d) to isolate in a convenient manner the carbon lost. The reason for failure of the Beckman monooxime degradation scheme is not readily apparent. Failure to form the tosylated intermediate may be one explanation. The Barbier-Wieland sequence, on the other hand, has given a satisfactory yield of the degradation products, the fatty acid ester (55.8%), and benzophenone (52.2%). 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Boston, Massachusetts 104 DL-Glucose-UL-14C Malonic-2—14C acid DL—Methionine-2-14C Sodium pyruvate-5-13C DL-Cysteine-BSS DL-Ornithine—2-14C DL-Serine-5—14C DL-Cystine-1-14C Pimelic-7-14C acid Sodium sulfate-355 APPENDIX 2 FORMULAE FOR COUNTING STATISTICS AND ERRORS (64, 65) Standard Error of an Observed Activity, n. 0 = (n) Standard Deviation of a Net Rate, RS. 0 = (Rs+b + 58> R = rate R tS tb s = sample b = backround t = time Optimal Counting Time Distribution. _t_s_ = (118%) 6 tb Rb Probable Standard Error of the Specific Activity, A. 02 62 0 = R + c Ii A (R2 C2 A C — concentration 3 P = 0.6745 0A 105