A Study of Some Naturally Occurring and Synthetic Cis-Trans Polyenes Citation Pinckard, James Howard (1950) A Study of Some Naturally Occurring and Synthetic Cis-Trans Polyenes. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/sbxe-af89. https://resolver.caltech.edu/CaltechTHESIS:06252025-223435637 Abstract No abstract. Item Type: Thesis (Dissertation (Ph.D.)) Subject Keywords: (Chemistry and Biology) Degree Grantor: California Institute of Technology Division: Chemistry and Chemical Engineering Major Option: Chemistry Minor Option: Biology Thesis Availability: Public (worldwide access) Research Advisor(s): Zechmeister, Laszlo Thesis Committee: Unknown, Unknown Defense Date: 1 January 1950 Record Number: CaltechTHESIS:06252025-223435637 Persistent URL: https://resolver.caltech.edu/CaltechTHESIS:06252025-223435637 DOI: 10.7907/sbxe-af89 Default Usage Policy: No commercial reproduction, distribution, display or performance rights in this work are provided. ID Code: 17487 Collection: CaltechTHESIS Deposited By: Benjamin Perez Deposited On: 27 Jun 2025 22:30 Last Modified: 27 Jun 2025 23:17 Thesis Files PDF - Final Version See Usage Policy. 38MB Repository Staff Only: item control page

A STUDY AND 01 SO ME WATURALLY OCCURRING SYNTHETIC CIS•TRAIS POLYENES Thesis by James Howard Pinckard In Partial J'ulflllment of the Requirements For the Degree ot Doctor of Philosophy California Instituw of Technology Pasadena, Calitornia 19-49 The author wishes to express his deep appreciation t:o Professor Zeehmeist.r ·ror his 1-tnest and gudanee in the probleru connected with this research. .The application of modern laboratory methods in stereochemistry is still in a relativ•lY early stage of development. Thus, although the structural study ot natural and synthetic polyenes has expanded in recent years, the stereochemical probl..ems presented by these nigbly unsaturated compounds are by no ineans completely solved. Indeed, tor a long time · there existed some doubt as to whether the presence of such a long, con• jugated double bond system would not prevent m-kw isomerism. The experimental evidence gathered in the study of carotenoids and synthetic polyenes in the last two decades leaves little doubt about the existence of m..KS isomers in such systems. Their study has further emphasized the idea that the morphology of molecules plays a great part in determining their properties, and that the effect ot stereochemical variations may even exceed that or structural alterations. The present study includes experiments on some physical and biological properties ot stereoisomeric polyenes and also on the isolation of a new colorless polyene whose stereochemical investigation has not yet been carried out. As will be shown, a combination or chromatographic and speetroscopio methods constitutes a very useful tool in this field. Tb• isolation ot sue new st.r•oi•~rs ot lyeopent is presented, and the proper\te, of..,,tJ:tese polY""!Ul'"'lfcopenes are correlated with. . . . . . ' ' . • .. . their ster1c configuration and in particular their s~ctral cmu-acter1st1os ~• discussed in the light of modern theories ot the natve of light_ absorption by carotenotda. On the basis of these theories tome pr.-dictions a1"$ made as to the :number ot .sk doubl• bonds and also about the ~eoted pt'Operties of p~l)-•W toras in A comparative stud;y or the t!u-ee stereouomerio : l,4•d1ph~l• butadieaes was c~ictd out; eu.ip1oying methods ot ohl'olnatographf and spectroaeopy, and aome po1nta ot similarity between th• a1>41ctral ohai'acteistio• or these synthetic pol7enee tUltl th• i1aturaU7 occur• ring carotenol- a,9 pointed out. fhe proTitalwl A aotivit7 ot SOM aJa isomers or Y •oarottm• was teated 1n feeding experimen~ using chioka as -.U q l"ats and :tt i s shown t h a t • .u,. i s o m e r may h a v • a p r o v i t u d a A a o t i v i t , 7 • • great •• 1-ta all•k!PI fora. A • - t oolotleaa pol7$1ie,te1111ed phywtluenol, was isolated and some qualitative o-ervatiens tJt its chemical and ipeotral oharaeterist1,es are deeeribed. Table it Cfttetf I. Isolation and Study ot Some Naturally Occurring .9b-Caroteno14 Pigmeate. • -'• Introduction •. • •• • • •••. • • , ....... . ... . . . ~ ...•·. •.•........... • 1 1. Steric Poss.1bilit1ea of the Carotenoiu ••••...••..•..• 1 2. Spectral Characteristics or Carotenoids •••.•.•.••..••• l.O 2. Some He:wSte~:t.aom•x-s of l,ycopene ....................... 16 3. SHQtral Gharaoteristies ot the Polrsk•lycopenes ..... 18 4. .ldeorpticm Characteristiea of the Poly-cia•l70openea •• 29 s. .au. I11omws in th♦ ¥' •carotene set........... •••••• ••• •• 29 c. J>olJ"-Ol••l,oopenes Occurring in the "fangeue• tomato ...... 3S D. '11- h°O'fitaldn A Potenc7 of Som• Stereouommo ¥ ..carotenes ..... • • . • • • • • • • . •r . .. • • • . . . • • • .. • . • . . • . • • . . .. • • • . . • .. 36 1. PrevioUI Wor-k on St~eolsomeric Prov1tam1ns A. ........... 36 2.. Confipration and B1o•potene7 1n the 'if -carotene Set •• 38 II. Phytotlwtnol.t a New, Batu:rall.7 Occurring Pol.yen• A. In.t-rocluotion ................ .......... ........... ,, •.••.•.••••• 41 B:. Charact.Joizati<m ot fbytotluenol ........................... 42 III. , StereotsQmetrtc l)iphenylbutadienea A.. lntrocluetion ....................................................... 4S 1.. Stereoisomeriam 1n Diphe117lpolyenea ....................... 4S 2.. SK and trlP@ Stilbenes .................................... 46 B.. Sterle Forms ot l,4•Diphenylbutadien•••·•• .......................... 48 l .. larlier investigations on Diphenylbutadiene .............. 48 2. A Comparative Study ot the Stereoisomeric 1,4-l>iphenylbutadienes ....................................... 48 •&itJ)er1nHtntal Part- A. Introduction ••.•••••.• •••••••••••• ~ ••••••••••.. ••.-•.·,. •• .• •••• 63 3. fo~•lJoopen•• l•lll •.•••..••••.•..••••••••• ••••· 69 4. Ul•Mau• '( -cai:rot.ne •••••••••••••••••••••••••• -~• • • 70 S. ie.o - "f •carotene f • ••..••••••••• .-••••••••• -. •• •.• ·• • • • • ?'1 C. Pol,...Gf;•l1cope11es hom the •'J!tUtg.-ine• Tomato......... • 81 D. Pr•parattoa of the Material Used tor Bioaeaaya •••••••••• 86 II. Golorleaa Pol,-.. A. lsolattoa ot Pb.ytot'lu•n.• t.roa Jacv,nclA.............. o . . . 92 B. holatioa of Pbyt(ltl.uen.ol t.roa Tomato Jaate ............. 94 Ill. 87l'lthes:la ot Ste:reo1eomeic Dipheqlbutaditn• A. \r•!!t•DipheDJlbuteniae •••••••••••••••••.• •• •• • • • • • • • • • • • • 99 B. Sk--kl!J•l>tphCH17lbutadiene •••••••••••••••••••••••••• •• 100 c. JH•!ll•Diphenylbutadieae............. ,. ••••••••• ,. • , •• ••. 102 IV'. References...... . . • . . . • . • • • • . • . . . . . . . . . • . • . . . . . . • . . • • . . . • .• • 106 I. ISOLATION AND STUDY OF SOJ.1E NATURALLY OCOUIUUNG !!l-~OTENOlD PIGMENTS A. INTRODUCTION 1. §i1ric t:2ss:U?w.t,,1 2f. l:!!! Qarottnoiy Without going into great detail, some of the reasons for believing that sou torms ot the carotenoids ~present true S!f•trana isomers should be given on a historical basis. the firet example of a stereoisomeric f'ornt ot a carotenoid was discovered in 1923 by He.rzig and Faltis (21), who were unable to account tor the chance appearance ot an unexpected form of biXin (C2;H3o()4)• latter and co-workers (2.3) made an analogous observation in 1929, and the opinion was advanced that this was a case ot .£i!•:\£ys isomerism s:f.nett the conversion of the naturally occurring ("labile") bix1n into the stable (•new") form took place upon the addition of catalytic amounts of iodine. Gillam and El Ridi observed the first case of isomerism in c40carotenoids in 1935 (15,16,17), and both dou'i>le bond migration and geometrical isome.r i'sm were discussed 'by them as possibil1 ties. Observations in other carotenoid set.s nre soon made (51,77,78) and in 1939 Zechmeister and Tuzson (79) introduced the use of iodine in the field ot 040-carot.enoids to catalyze sterie changes. As work progressed, Zechmeister and co-workers (S2,53,60,79) accumulated increased evidence tor them• llW character ot a num.ber of phenomena. The initial, tentative assumption of double bond migrations had to be abandoned on the tollowing basis. a) The observed reversible changes are spontaneous in many instances or are effected under very mild conditions. b) Iodine, which, since Anschutz•s work (1), became a well• known catalyst for w,•trys rearrangements, is effective in the tield of the caroteno1ds. c) Some earotenoid pigments., whose ehromophore is blocked on both ends by conjugated keto groups (53), show the same phenomenon of a reversible isomerism, although double bond migration aould not take place here unless this migration could be into some of the met.h;yl side chains, d) The large num'beJ' of isomers obsex-ve<l in some cases, t,.g. p ,..carotene (65), cannot be aoeounted tor by any- double bond migration proces$es. e) If double bond migration oceurred, an easy conversion of c,{,•carotene (C4off56) to (3 •caroten,e (G4oHs6) and of lutein (C401fs6f>2) to zeaxanthin (C4of150<>2) would be expected to take place. Such conversions bave never been observed under the conditions whieh cause isomerism. Granted that we are dealing with ~trang isomerism in. this field, the possible scope of res~arch still appears to be very extended. If all the double bonds ill such a mole.c ule as l7eopue (C4ol56) 1 which has eleven conjugated and two isolated double bonds, could undergo m•trans rotations, the number of possible isomers would l.MI c,yer a thtuaand. However, according to Pauling (4.3,59), we can place drastic limitations on this nUlllber by a consideration or the geometry or the molecul•• In such long conjugated systems of e.a rbon•carbon double bonds, the chromophore will be essentially coplanar as a result of the partial double bond. character of, the single bonds. In an unbranched p0lyene ohromophore, each double bond could exist in a a!l configuration (a alight ster1c hindrance would be present), but, 1n the earotenoids, which have a chromophore consisting of dehydrogenat.ed isoprene units, only o:ne double bond in each C5•unit is spatially unhindered, and very probably only this type ot double bond could have a .£ll configuration. This steric hindrance is due to the oonfiict between a methyl group and a hydrogen atOl!l when the C•methyl r~e l. Drawtnss showing overlapping of hydrogen atoms in --Cll•QR•CR--Oll• and ot hy@oger1 and methyl in -QH--GB.sel•OOU.3 wlth SI configuration {fa.uling, 60). group is adjacent to a_su double bond (Figure l). Since this conflict is considerable; Pauling concluded that this type of double bond would ht •stereochemically ineffective•. Other types excluded from this discussion are those double bonds in the cyclohexene ring a,nd also double bonds carrying a C•dimethyl group. There are several known instances where relatively stable cis•isomers occur in spite of a marked spatial conflict (34,47,24) but these are all structures containing a shorter conjugated system in which ooplanarity is more easily disturbed. Objections hav• been raised to this general concept or steric hindrance by Karrer (25,24) 1n the cases of bixin and ~ •methyl muconic acid (c.,a8o4 ), but, since the final evidence of an absolute structure determination is lacking for any stereoisomeric carotenoid, the interpretation of the results obtained in the present work will be based on the theory that double bonds of the type •CH=-ica-p cannot assume Sll configuration in such long conjugated systems. CH:3 tormulas have been given (.3.2,60) for calculating the number of possible stereoisomers in polyenes by Kuhn and Winterstein and also Pauling. Two subclasses are recognized; the unsymmetrical. and symmetrical. For unsymmetrical molecules, whose two halves are different, the number . n ct stereoisomers, N, would be N = 2, where n is . the number of ster•oehemir . 1 effective double bonds. For symmetrical mol.ecules with n odd, n•l I• l n • N: 2 • • (2 • ♦ l) and tor n even, N : r" • (iJ. + 1). ln the 21 ¥ case of l10opene, with seven stereochemicaUy effective double bonds (Figure 2), the number of predicted isomers is seventy-two. Skeleton models of the non•hindered stereoisomerio lyoopenes (50) are given in figure 3. The distribution of lycopene stereoisomers according to the ~I J 1 H l ¢H3 Ii s i :Z. H B 2 U J.1 B HB ohemically effective double bonds numbered* Figure 2 • . All-trans l;vcopene with the seven stere&- CH.3 H The letters following A in the alphabet designate 18omer11 If a pigment also yields stereoisomers whioh ar. ada&rbed above the all-trang for.m, those with increased affinity~• designated as neo T1 U, v. etc. There are some inconsistencies in particule:r cases' which arose on a historical basis .. In addition., the term poly-;G§ (62) applied toa member 0£ a given carotenoid set indicates that half or more of its ster-®1-mieal.17 etfect.ive double bonds have the !ii configuration., the term •pro11 ha$ been used. to 4-sipate a naturally occurring neo form of a oarotenoid with Ul8Jt1' sJ&_double bonds,= £orexample, prolycopene. with weaker adsorbability than A. example, neolycopene A. from either end or the chromophore. terminal group. In a symmetrical moleoule, · evidently the numbering may~· started Because the -0onfigurations of 18.omers are. still unknown 1n most instances i t is eustomary to employ the prefix •neo• ,- for bond of theeonJugated system will be assigned. an ~iciZ§d number to avoid con• ~ion with th• numbering ot carbon atoms. The lowest number will be giv♦n to the double bond in the (3 •ionone ring, or, i f th$ double bond of this ring is not part of the chromophore, to the conjugated bond nearest th• (3•ionone r:ing~ In the absence of such a system, an ll(. •ionone ring receives preference over an aliphatic * The follOWing nomenele.ture was proposed by Zeehmeister and Polgar (68). Eaeh double Gl.3 1l H Cl3 B llJI i H f X 11 B QH3 lJ 9B3 H ~Ii3 JBJ. B-A.,Jly, c'. -...,,;-c,f; .'c¢ '-r;«"91-1;,,/\;,;,c'v~c,,,i\;r-,/(' '-r;~ 'on3 B t 't X I II m IX Skeleton models of the seventy•two sterieal.11 unh:lnc!W.•d ,su•tr,g.ng isomeric forms of lycopene. The shgl.• •14• lin•s r•present methyl groupe; the black circl•s stand f'or th• carbon skeleton of the end groups in the lycopene molecule~ xxvm )= xxx11 xxx111 ~xxx1v XXXV XXXVI 7iguN 3 .. Continu$d XL'\ZI XXXVII I i xxxvur :XL'\ZII : XXXIX XLIZ · L LI XLI[ Lil XUS7 LII1 XLV LN LXN L'\ZI LIX LZSZI!l LX LXIX LXI LXIT LXIII figu.i,• 3 • Ooncluded number or .s,a double bonds is given in Table I. Table I Distribution of the Sterieally Unhindered Lycopene Isomers According to Number of ci; Double Bonds Type or isomer Number of isomers all•trans 1 tnono•&i!, 4 12 19 19 12 hexa•s.!!· 4 hepta•si§. l 2. Spectral Chru:'aeteristies st Garotenoid! Since much of the present knowledge a.bout the number and arrangement of cis double bonds in carotenoids :ls based on a study of their light absorption• a brief' discussion of some theoretical and experimental results obtained by earlier authors in this field will be given (49,43,59). That member of a caro·tenoid stereoisomeric set* which has the maximum light absorption in the fundamental band is believed to be the all•tran@ member or the set. This assumption is reasonable si nce the all~trans form would be the most coplanar of all possible steric forms. Even in t hose * The term "Stereoisomeri c set•• includes all c;ts-trans isomers of a given carotenoid. -11- double bonds which are classed as stereoehemically effective, the sil con• figuration would involve a slight spatial conflict bet\veen the hydrogen atoms on the carbon atoms a.djaeent to the 9:ia bond (43)1 Figure l. This conflict would involve a slight rotation out of coplanarity and inter• ference with complete conjugation would eause the absorption maximum to shift toward shorter wave lengths. EJ..-perim.ental evidence supports this explanation since it is gen~rally observed that 2:1.s isomers have their position of maximum light absorption. shifted toward shorter wave lengths as compared with the all-~r~ form, and that the absorption bands of the / all-trans member of the set shift to shorter wave lengths men stereoisomerization occUl"s.* This shift in wave length of' the absorption ·bands is the basis from which we can estimate the number of ais double bonds. It has been .found experimentally, by Zechmeister .ll !.l• (49), t hat, in a great many earotenoid sets, the smallest decrease in the position of the longest wave length maximum (visually determined in the Zeiss spectroscope) is near 4 to; mu.. This may very well be the effect due to a single tran§ m rotation. furthermore, values for this dec~ease in other 9is-isomers are approximately integral multiples of this value. Small deviations can reasonably be explained by the still unexplored effect of the position of the cis bond (or bonds) in the chromophore, since a. peripheral~ double bond will not be expected to have the same int erference with resonance as one which is centrally located. Earlier studies by Zechmeister and Polg'1- (66) of the absorption * One exception to this statement has been reported in the case of neo- vitamin A (44,37) which cannot yet be discussed in a satisf'actor-.f manner because of the scarcity of experimental data. spectra ot carotenoids showed the presence of a band 1n the near ultra• violet region. 'fhe wave length .di.fterenee between this new peak, termed the •cis•peaJttt, and the longest wavelength maximum is nearly constant (1.42 ,a) tor all c40-carotenoids tested so ta:r .. A theoretical interpretation ot this effect has been given by Paulilag !i Al• 09) wbiQh permits the approximate location o:f.' .s!J. double bonds in some members of a stereoisomeric set. A qualitative discussion of the theory ot light absorption, as applied specifically to lycopene {Pauliilg and co-workers) (59), has been given, and may be applied generally for any set of carotenoid stereoisomers. According to these authors we may picture a vibration of the mobile 11 unsature.tion• electrons along the conjugated system of double bonds, and the following classical modes or vibration may be correlated with certain bands in the absorption spectra. a) The electl-ons concentrate fir,s t near one end then near the other end ot the conjUgated system. This mode of vibration is correlated with the .t'tuldamental absorption band. b) The electrons move from the two ends or the conju.gated s79tem toward the middle and f~om the middle toward the two ends. This oscillation of charge would cottespond to the "s11•peak1 region or rust overtone. c) 'the electrons concentrate alternately in the first and third and then the second and fourth quarters of the conjugated system. This mode ot vibration would correspond to the second overtone absorption band. The intensity or an absorption band is proportional to the square of the dipole moment and, thus, essentially to the square of the length -13• of the conjugated system (43.,38,39). Since the molecules 0£ all isomers have a shorter distance between the ends of the chromophore than the aJ.l•trans form, the intensity of the fundamental band of the cis-isomer should be less than that of ·the ~-trans meraber of the set. The decrease in intensity of the .fu.nda.menta.1 band of a 2is-iso:.1er was noted by Zechmeister and Tuzson (78,79) for neolycopene A, and a theoretical interpretation of' this effect was subsequently given by Pauling (43). The nature of the vibratio:;.1 which gives rise to the 11 gis•peak" is such that ii' ·the cb1·omophore is syillmetrical abou·l; a center, there is no dipole moment and thus no absorption in the region of the first overtone. All ste:raoisomers without this center of symmetry should have some light absorption in this region, but the inwnsity of this band would ·be of appreciable magnitude only for those, such as ,2-mono•Ci§•lycopene, which have a. ·bend near the middle of the chromophore. The intensity of the Ci§•peak is estimated to be roughly proportional to the square of the distance between the center of the conjugated system and the mid-point o:f' the straight line between its two ends (Mulliken 38, 39; Fauling 59). A sumraary of the stereochemice.l diagnosis (49) of a given carotenoid can be based, to a considerable ext.ent, on the observed spectral data as demonstrated in Table II. ..14. Table :tI Character or the $Xtinction Curves for .Some Types ot Carotenoid Stereoisomers Stereoisomeric • type Change of extinction attar Shapo ot the extinction curve in the m•peak !:!SUP . . Iodine oqa1zs1, ln the .aJa• In the funda• . megtal bapd PHi .£te;itA decrease increase peak increase decrease fl.at targe increase increase aU-:tetAD, . . I -15- B. POLY-Cis-LICOPENES OCCURRING IN THE :F'RUl'l' OIi' .fYRAOAN'l'§A It \"las discovered earlier by several investigators (Went, Zechmeister, LeRosen; Schroedex-, Escue; 60,;6,76,54,75,33) that several plant materials contained prolycopene and pro~~ •carotene; and that these two pigments are polY,,.Ja! isomers 0£ the ordinary all•trans•lyoopene and all-tra.n@• 'o •carotene. The presence of several other poly-,!!! isomers of lycopene was also indicated by the speetral data of some pigments observtd on the ehromatographic column. The isolation of some of these new ~-isomers is described in the following section. l. Composition~~ _tu:agantha pigment The main bulk of the carotenoids in the ripe berries of PYraeantha angustifo;i.ia Scbneid. consists of hydrocarbons (76) including some members of the ~ -carotene, /3 •carotene, ~-carotene and lycopene sets (Table III), as well as the eolorless polyene, phytofluene (72). In addition to some stereoisomeric lycopenes and ~-carotenes, a well known isomer of f, •carotene, neo• f -carotene U, WB.S always observed on t he chromatographic column. W ith reference to the latter pigment, it must be stressed that if a stereoisomer is easily produced from the all-trans molecule by methods similar to those used in the isolation process, then a natural occurrence of this isomer should not be claimed. However, another crystallizable .21§. pigment, termed neo• ~ •carotene P, was observed as a constituent of stereoisomeric mixtures as obtained from all-trans- ~ •carotene. 'l'able III List of some Polyene Hydrocarbons Isolated from fYI"e.cantha Berries (In the order of decreasing adsorption affinities on calcium hydroxide; petroleum ether, Oto 8% acetone. The figures denote amounts isolated per l kg. or fresh berries). All•traps•lycopene l4 mg. Poly•s!l•lycopene I 3 Poly•S!•lycopene II o.s P0ly•_m•lycopene III 1.2 All•tra.ns• 'l5 •carotene 3 }leo- 'lS -earotene P 3 :Pr.o lycopene 46 Poly•eis•lycopene IV about 0.05 Poly-£iJ!•lycopene V about 0.05 Foly-~•lycopene VI a.bout 0.05 Pro- ~ -carotene 2. 13 Some New §ter9oisoiqera 2!, Lycqpey As listed in Table III, six new isomers were found and termed polycis•lyeopenes I to VI in order of their decreasing adsorbabilities, in petroleum ether solution, on lime. Poly•gis-lycopenes l, II and III were isolated in the form of analytically pure cryst,als (Figures 24, 25). They are accepted as true constituents of Pyracantha for the following reasons: (a) When any one of them was refluxed ·in pet,roleum ether solution, under carbon dioxide for half an hour, a subsequent chromatogram showed that only traces of other isomers were formed, and their spectral, curves, after re.fluxing, showed no appreciable change. (b) Prolycopene, from which these three pigments might arise, as a result of stereo• isomerization du.ring the isolation processes, is also thermostable under the conditions just given and is not likely to be altered greatly .under the conditions used for the isolation of the pi gments . (c) No poly•oj_s• lycopene has ever been obtained after heating solutions of all-trans• lycopene. The poly-s!§.-lycopenes IV, V, and VI were observed only in solution and their total quantity was small. The poly•cis-lycopenes I•VI were accepted as belonging to the lycop.ene set on the i'ollowing basis. (a) When their petroleum ether solutions are examined in the visual spectroscope, rather blurred bands or shadowed areas are evident. However , upon the addition Qf a drop of dilute iodine solution to the spectroscopic cell, the bands of the stereoisomeric lycopene equilibrium mixture (5021 471, 441.5 mu) appear almost instantly. There is a great increase in the intensity and sharpness of the bands. (b) Each of these stereoisomers was catalyzed with iodine and chroma.to• g-caphed. The main pigment zone was s~parated from the others by cutting and a mixed chromatogram test performed using an authentic sample of alltrans•lycopene. There was no separation, · on 't he chromatographic column, of the main pigment obtained from any of the six and fil•trans•lycopene. (c) The spectral curve obtained from each of the six poly•.$!! isomers, after iodine catalysis, proved to be practically identical with the corresponding curve of iodine catalyzed all-~•lycopene or prolycopene (* ngures 4•10 and Table X). (d) ihe analytical data (fable IV) indicate the same chemical composition. Carbon and Hydrogen Valtles round for Some Crystalline J>olyene Hydroearbone s Py:rac@!ltlJ.a (Cal.ad. £or C4oH.s6' C., .89.48; H 10.52) Pigment Hydrogen (j) Qarbon ($) . Prolyeopent 89.02 . · 89.62 10.67 folr-cu•lftopene J: . a,.04,ff 89.ll ,10.56** 11.03 Poly-cis-~opene XI • 89.34 . 10.,5 · 10.59 All•tra,ns• ?S•ea:toten•. ff 89.20"' · 89.49 u.01. O•earotene P. 89.18 10.57 Pro• 1S -carotene. . 89. 56 10.92 Neo- 10.87 10.58 10.58 3. Spectral Oharacterietics '.!!£ ,:2 Polz•c\p•lyoop4Pl•! As mentioned .earlier,. it has been obs&l"Ved, in all stereoisomerie caroteno:l.d sets tested so far, that each tr,mg ~ ,w shift causes a a.iain de~rease in the wave length position ot the maximum of the * ln all apeotral curves which follow, th• term Er!;. is defined as 1 • log _!,! where t, is the length 0£ the solution in L• o I the light path; c is the concentration in mQles per liter; and Io and I are the initial and final light 1nten$itiea (66). Lis expressed in om. equal. to "These values have been corrected tor about l$ ash. The smallest such decrease known (59) for funda."l!.ental absorption band. a member of the stereoisomertc lyeopene set is 7. 5 mu, in hexa.'i'le, for neojyeopene A {determined in the Beckman spectrophotometer). -~1/( If this value is ass'Wlled to be that whieh corresponds to a single tranp ~ .s!i shift, the probable number of cis bonds can be es•i;:i.ma.ted foI' some members of the lyeopene set (Table V). Table V FrQba.ble Number of ili Double Bonds of Some Stereo• isomeric Lycopenes; Based on Extinction Data i..11 Hexane (The pigments are .J.isted in the sequence of decreasing wave lengths of Amax. and increasing number or !ii double bonds probably present) Diff • . in Member of the l max. (mjl) set = Amax. Emol. from the all1 cm. form kman spectra- X 10• 4 photometer) at ~max. .. (mµ) Relative Extinction areas between 320 and 560 mµ Probable Number of.!11 Double Bonds Preast . All.. trans 472-473 0 18.6 100 Neo A 465 7.5 12. 2 82 1 444-445 28 12.3 76 4- 5 Poly•S,!! llI 444•445 28 11.3 7l 4•5 Pol:r•eis II /4Al 31.5 11,4 70 4... 5 Prolycopene 438 34.5 10.3 60 5...6 Pe>ly•cis VI 433 39. 5 8.1 54 6 41 9.0 52 6 46.5 10.4 62 6-7 Poly-~ I Poly• cis V 431-432 Poly•eis IV /4.26 ' L-- I '-- - ~ I- I-- L-- ' I ' ' I I '-"/ I .......... I I I .._,, I I / I I I \ \ \ \ \ \ I \ 300 I -/ /\ I / I / / ' I /-..,/ I \ ," I \ I - ' I I / I 1_ I I I I I I I - I 400 I I. / I ~ I -- - - -- +--- - · ,- - ' I I I I I I I I I I I - I - I I I - I I f\ _ I - I I - -- - - - I I I I I I '- I I- I_ ,I I - 1- I I 1I I - ,- \ I I I - - - - 500mu - - I (\ I I i I I I / I I I I I 0 11- I I -~ I \ I I I'I I - I I I /'\JI I Molecular extinction curves or poly- cis•lycopene I in hexane: - - , fresh solution; •----, after iodine catalysis, in light. •• I ~ I Figure 4 . 5 _JE b s;t- ?< . Oo E_ w - 10 . - 1-- ' ,__ ~ ' ' ? fl.) w ~- _j 00 5 e X 'o o;;t 10 Figure 5. I -' - (\ 300 / I I _400 \ I \ I I \ J \ \ \ \ I \ '' ' ' I I I I 'I I 500mu \ I '\ - II I I \ - \ - '' \ \ \ I l!lfoleeul.ar extin-etion curves of poly•!!!,•lycopene II in hexanea: . - , · fresh solution; --- , after iodine catalysis• in light. • \ \ I \ I \ ," I \ t ~ t I- ._ - l l ' l .....__..,,~ I I \ \\ 1 I I ,- \---+- 300 I \ '------l--- I \ --- -~ ~; I I II /I I ~- I / I I \ A I \ I / I 400 I / I , I I I I I I I I I I , I I I I (\ I \ \ I I I I ' V - -- - - I I I I I ', 'I I 'I - ~~ \ ,'I - I I I I ~ ' '' - I I - - - 5OOrn,u I I I \ I \ \ - I I I ''~ I ,'\ I I \ \ I \ •--, af'ter iodine ca~ysis, in light. Molecular extinct.ion curves of poly-ci;•lyeopene III in hexanes - , fresh solution; '""' Figure 6. ._ I- - I- I I I I I I I I I I I ,"v +--+---+---+---+---+- II 5 t---+--+--+-+-+-+--+---+--+-- _jE 0o E_ w x. b <;t 1 0 - - - - -- ._ I- I- f I I/\ I I I I I I I I I I 10 \ \ \ \ ,_, \ /\\ \ I \\., I I _jE I I I\ I Oo I I I- I r\ I I I I- I I I ','\ I 5 I I I\ ' \ \ \ ,....," ' , -/ / \,,. 'I '' I I \ __, I \ \ I \ / I I\ \ I ,_., I /... ,_...' 300 Figur• 7. - I I \ I \ \ 400 ,_ I I- ,- 0 X \ \ ' I- I i W \ \ I ,;;t- E- ~ \ ~ Molecular extincti on curves of pol y-sa- lycopene IV in hexa.nes , fresh solution;-- -- , after iodine catal.ysis, in light. 500mµ /\ I ' -I \ , l , I I I I /'.J I I I 10 I 'I\ I '>--I\ \ I \ I ',-, ',_ \ I I I ,- \ I Cl V 'II _ ',_ I ,I I I ,- I /_ I '' I - I '',_ \ ''I ' I I 5 i\ I I - I (\ I \ I \ \ I I\ \ I \ ~ \ ~,J_ "~ \ I ,., I f\ I ,- I \ I r-' \ I I I -,..., I -/ I 300 Figure 8. I 400 Molecular extinction curves of poly-s!!-l;rcopene V in hexane: - -·,_ fresh solution; ••-••, after iodint catalysis, in light. 500mu --- - 1---l-- ~ - -------+- - + - - + - -- -1---- -+-- -+-- --+--- /\ -+-- -+----+- - - - - - l - ~ , --+----+-- - + ---+------l-- , - I +----- I / \ \ -+----+---- I I I I ' \ I\ \ I \ \_/ \ - r..J IO ---+--~ \ I \ \ I \ " , - \ I 1----+---l---l------l-----+------+-+-----+-----+-----l- ' I ~ - - + - - - - - - - 4 - - - - - + -- I - + -------4~ - - + - - - + - - - f - - - - - + - - - / \- / <;t \ ,- I ., 1-~ I '1 I 0 X I _jE Oo l--+--------1---1-E_ -1---+--- - + - ----+----+-------+-- w \- ',- I I l-----1---- / I -+-----+--- 5 I f\ I I\ \ I \ \ ~ \.......... ~ ,✓ / - \ \ I+---+----+---+-- - - + - - - - - - l - - I\ - \ I '\ ___,___ I I \ -~/ I I _ __,___ ___,___ 'I - / I rJ \ / ' -\ / - - - + -- -+----+----+---, ._/ \ ~ - ~ - - + - - - + - , / - - + - - - + - - - - + - - - - - - l- - + - -+ - --+----+-- I 300 Figur$ 9 . 400 Molecular extinction eu.rvee oi' poly• cis- lyeopene VI i n hexanei .- -, fresh s.o lution; .;... _.,..;. , after iodine .c atalysis, in l ight. • 500mµ. c:::. 0 LO ~-o,xwo '-:i v ·1ow .::i ----------- - 4 strict proportionality between the number of s!I bonds and the value ot the spectral :,h:1tt is not to be expect~, but the estimates in fable V - should'" close $pproxime.tions ,to the correct number (perhaps ♦ ones&! double bond). fhe pol.y-eis•l;ycopenes from .f:vDoa.gtwa have four to seven Jll bonds out or the seven sterically unhindered double bonds. The intensity of the tundamenta.l band for each or the stereoisomerio poly-,9'1•lycopenes is considerably less than that or the all•tran, form. Adopting the suggestion made by PauJ.ing (59), the area of the extinction curves 1s taken as a measure of the intensity of light absorption. The areas under the curves, from .320 tc:> 560 mp for ea.oh of the poly-mfOX'DlS, were measured with a planimeter, and these values were compared to the corresponding area of all•~•lycopene (fable V). In the theory developed to explain the spectral characteristics ot the lycopene set (59), it was pointed out that all isomers with a vertical plane of symmetry have a distance between the ends of the con• jugated system smaller than the all•trane form by a factor or cos o( , with c(: 27° 22' if the carbon-carbon bond angle along the chain is 125° 16 • • Since the inte~si ty or light absorption is proportional to the square of the dipole momeat and, thus, to the square of the distanc• between the two ends or the conjugated system, all isomers ot this type sho'Ul.d have intensities approximately 80 pff cent as great as the all• H'M!· form (cos2 27° 22 1 : tv0.8). All e>ther isomers would be expect•d to show intensities between 80 and 100 per cent ot that ot all-gans1.ycopene (59). We notice from Table V that the smallest area !or a'AY' of the poly-au. forms amounts to about 50 per cent ot the area covered b7 the curve of all-~•lycopene, and that all or the poly•siJ! forms have areas less than 80 per cent of the all•~ form. The lesser intensity- of the fundamental band of poly-$k•lycopenes becomes evident on simple inspection of either a t1olution or adsorbate. All•W:Mf•lycopene solutions are intensely red while a stereoisomer such as poly-.w,•lycopene VX appears to be yellow at similar concentrations. The de<:rease 1n tine structure of the fundamental band of an all- :t,;:w oarot.noid is .partie1.Uarly conspicuous when the number of s1I double bonds increases. Steric forms . with one peripheraily located s.u double bond such as neo- d-i •caro.tene U or neo- ,t3 -carotene U eho• a moderately decreased extinction without mueh alteration in the relative heights ot the peaks which defines the degree or tine structure. A central mono-su. isomer such as neolycopene A shows somewhat more ot a change. Th♦ rat1os or the extinction values at the two longest wa11• length maxima and the intermediate minimlllll are 1.45 al 1 1.7'/ for neolycopene A; while for all•tr§Ql•lycopene the corresponding values are 1.72 s ls l.86J and for poly-4'1 lycopene II, l.-07 a 1 1 1.12. In the spectral curve of poly•$•1,copene VI (Figure 9) there is scarcely an indication of fine structure. An eaination of the models in figure 3 shows that most of the isomers with three or more SJ. double bonds are characterized by an overall linear shape ot their molecules. Such a rod•like shape is also characteristic tor those isomers whieb have one peripherally located Sia double bond or two adjacent m double 'bonds. No Jil..peak (in the lycopene set, at around 360 lllU) is to be expected for isomers ot this typ~. The ,· same statement is valid tor all seven poly•s!l•lyeopenes Jmon at the present time (including prolyeopene). No !a§. peak appears in the region between 320 and JSO ll}1 in any instance for these sterio fOl"J.UJ. The region of the. second overtone, which is located near 295 mu in the case of all•tt:Ma•lycopene• is more dilt'ioult to interpret in the OUl"Ves of its pol.y•.dl isomers. It the second overtone band has sb.if'ted to shorter wav"1engtb, as the fundamental band has shifted, then it may be represented by the peaks near 255 or 235 mp.. These peaks show a decrease 1n height (compared to the 295 mp peak or the all•;irJm! torm) which pal"allels the decrease in height of the fundamental band or the poly•.9U torma as compared to that ot all•~•lycopene. 4. J\dsorption Oharacter1stios !£ the ..l'Qll•c,s..lzco1:enee Although the exact relationship between adsorption affinities and stereochemieal contiguration is unknown, some general statements ean be made. It was shown earlier (78,79) that the mono-s isomer, neolycopene .A, had a moderate decrease in adsorption a.i"tinity on a lime column as ooJDpal'ed to the all•»-191 compoun.tl; in ligroin solution. All pol7•4U isomers known at the present time show much less adsorption af'.finity than the corr♦sponding all-trans form; furthermore the individual ditterencea between the poly-.9i1 forms are much sma.Uer than the d:tfterence between the all-tans form and any poly-Si!, compound. One can say, as an approx!Jnation, that the adsorbabllities decrease in the same ord•r as the extinction values ot the fundamental band. Figure 11 shows th• position and shape ot the top section of the respective fundamental. bands and demonstrates at the same ti.me the approximate adsorption sequence. The ettect of the accumulation of£&! bonds on the adsorption affinity is so great that some poly-!14-lycopene.s are adsorbed below all•trwim-~-carotene. s. ~ isomers ~ the .'6. -carotene set In con'trast to the large number or lycopene isomers in t:vraca.aiQI, the ~-carotene set seems to be represented only by three of its members; all'"'UIBI• i-carotene, pro• ~ •carotene, and a new isomer termed neo- K-~teneP. The absorption curve or neo• '6--carotene P (Figure 12) indicates that it 1, a monO•Ja isomer; and since no marked W•Pfilak is present the S!a bond is p.toba~y located near the end of t .b e chromophore. Its slightly dec~~d adsorption affinity on the lime column as eoJ!lP&red to th• all• trans form ie ·i n ageement with the behavior of co:rrespollding lycopene :tsomei-& • .Keo• ~•carotene U', which was described earlier (69), 1s adsorbed above the all•k!ms isomer and ispre21U111$bl;y also a .p$r1pheral mono-.swt iSlomel' • Since neo• l7{ •08%'ote1:1.e tJ and neo-- f •carotene U ar• both adsorbed above their aU•trang forms, this '-ncr.,ased adsorption affinity ot some steric forms, having peripheral . ,gu double bonds, ila7' be related to the presence or ring structures at the ends or 'the carotene chromophores. the possibility arises that neo- ¥ •carotene U has the $U bond near the hydroarOllatie ring while, in neo- 2S'-carotene P, th• .s! double bond could be located near the aliphatic end of the molecule. this would be in accordance with o·bservations in lycopene, which has a compltftely open chain structure and, all of whose known stereoisomers show decrease<l adsorption affinity in comparison to the aU-m.wi .form. The extinction curves of all•tra,ns- ~ •carotene {Figure 13} and pro~ •c,erotene (Figure 14) agree in all main features with curves published earliei- ( 59). Some results of bioassa,s carried out with all-trans• 't •earot•e and its two stereoisomers, pro• ~-carotene and nee,~ -~otene f are given in fart I, Sect.ion D. ALL-TRANS 15 12 I \ \ Il \fil 9 PROLYCOPENE 400 Figure 11. 450 500mJJ,. Molecular extinction eu.rvea of all-trans•lyeopene, prolyeopene and poly•ci~• lycopenes I • VI at their main m.."'l.Xima , in hexane. /·\ I \ I . \ I ~ \ I l - '/ 10 I -\ \ L-- ,- ,"'\ \..J \ I \ \ \ I I \ \ \ I I I ~ I 1I I I I \ \ I \ I \ I 5 \ \ I I \ I \ \ I I ,,,-, I 1---L, - V I "'\ '\ \ / \ \ I \ \ I / - ......... / I '\ '\ I / -..;:::/ 500mu. \. I I 300 400 ¥' figure 12. Molecular extinction curves ot neo• •carotene 1 . 1n hexane, ·. , ·fresh solutionj •• ;.. •• , afi.t" iodbat catalysis, in light~ \_ • ' /\ ~~ \ \ I t ;, \ 10 l - >- \ \ I I- \ '-/ r./ +- - I I \ \ \ I \ l I I I I \ I I 1 \ I. \ /J 5- I I I \ I ! I /2 I. I I I I r, I~~ /~ 1// / / "'-_, / / I I - - C - ~- I I I ~-1/ ......_..,,, ~ I I \ I \ I i \ I .___ i ~ t---- '\\ - \. 300 Figure 13. 500mµ. 400 Molecular extinction curves of all•tran1• ¥ •carot~n• in hexane: - - , fresh solution; - -..-, after iodue catalyais, in l i ght . : ,- 0 ~ (..) v 'o 5 I I --- Figure 14. - , _____ - - - ,- X _j ~w - 10 I- ,- - ,- I \ \ I \ \. I 300 I \ I I / / ~ / I ~ ' I I, I :.. . I I I 400 I I I I I I I I I I I I I I I I I I I I r-1 -- I I I I I I I I \ \ \ \ I\ - I I I ~ V I \ \ \ \ \ I I - - I \ \ ' \- \_ \ - - '',_ I '\ _ \ \\ - - - - - ••• 500mJJ, I \ ''I '' \ \ I '' \ I' - - - r • carotene in hexane, ....._, fresh solution; I I ••-.-, after iodine cataly~l!, Jn li~t.. ": Molecular extinction curves of pro-,. / / /"- --- I w I ~ C. NlLJ-Qu...l,ICOPENES OCCURRING IN THE "TANGERINE" TOJ:AATO 4t first in'Vestigation of the pigments in the "tangerine" variety- ot tomato (56) had indicated the presence of a considerable number ot stereoisomel'ic lycopenes, but that early study had to be confined to the main pigment, prolyeopene. An e:t"fort to isolate some of these minor isomers has now succeeded. ln addition to the main. pigment, prolycopene, two other poly•.sil•lycopenes wwe isolated in the £0"1 ot analytically pure crystals. These pigments proved to bt(l identical with the poly-cis•lycopenes I and Ill (& lml&• ca.ntha). The iaolation ot these two forms from a different plant material ("tangerine• tomato) is a confinnation of the earlier data obtained with tyrasgt}a,. especially since the spectroscopic characteristics 0£ these two compounds were found to be the same in samples from the two source materials. -.36- D. THE FROVITAMm A POTENCY OF SOtlE STERE0IS0MERIC Y-cAROTEl~ l. frev:i.oY§ Worf .2n Stereoisomeric Prov;tamin§ A The relation between spatial configuration and provitamin A activity of some carotenoids has been the object of several earlier studies. Qualitative work ws., carried out first by Gillam and co- workers (16,17) tor en unident:l.fied stereoisomer of {!' • carotene. They concluded that, in the rat, th~ vitamin A potency of the new steric :form was of the same order of magnitude as that of all•t,rarui- %-carotene itself. Kemi-nerer and Fraps (14,27, 28) later tested the same stereoisomer and found a potency one•half' that of f3 - carotene. A series of investigations by Deuel , Zechmeister and group (6,7, 8, 9, 10,ll) have covered a number of memb8rs in the stereoisomeric o( .. , fJ -, and c • carotene sets as well as in. the cryptoxanthin set. rhe Qha.nge in provitand.n A potency when an all- trans earotenoid undergoes one or more ;t,rans~ ci§ rearrangements ie a general phenomenon. If' complete information were available on the bio-activity of the possible stereoisomers of vitamin A, * a more definite choice could be made between possible reasons f or this change in potency. In accordance with the the~ry developed (43) about the number of stereochemically effective double bonds , the probable number of (non-hindered) steric forms of vitamin A would be f'our . So far, only one cis• trane isomer of vitamin A {neovitamin A) has been reported (44), and a preliminary test indicated that its potency 1n the rat is roughly equivalent to that of vitamin A itself. * "Vitamin Alt in this thesis should be interpreted as referring to vitamin Ai. •37- Among the possible reasons for a biopotency differing from that or the all•trw carotenoid are: a) The degree of sterie stability and chemical. resistance of the provitamin A or vitamin A isomers in the body. b) Solubility or adsorption differences. c) Specificity of enzyme systems for particular configurations. d) Various requirements for different species of animals . Some values obtained in this laboratory £or biopotencies of isomeric provitamins A in the rat are given in Table VI.. Table VI Relative Provitamin A Activities of Some 0arotenoids in the Rat Relative potency Literatl.ll'e (i> R~:ferenc1 All•trans• f-, •carotene 100 - Neo- (3 •carotene U 38 (8) Meo- fs•Carotene B 53 (6) All-trap;- o{ •carotene 53 (ll) Neo• o( •carotene U 13 (ll) Neo- o( -carotene B 16 (6) All•try.ns- .~ -carotene 28 (7) Neo• 2) •carotenes (mu:t~e) 16 this Thesis Pro.,. ~ •carotene 44 (7) o •carotene l 19 this Thesis All-,:trAA@•Cryptoxanthi:n 57 (9) Neocryptoxanthin A 42 (10) Substance Neo- 2. Configura;t:ion ,iW! Jl!O•PQt,ng:y J:s ~ b' -caro\tmg ~ .Kuhn and Brockmann (29) f;irst reported the provita.min A activity ot a ~ -carotene sample isolated from commercial earotene. Their experiments With rats. (30) gave a value of approximately 50% as eompar$d to p'•carotene. An investigation by Deuel, Zeehmeister ~ .il• (7) using ~ •carotene S m.mul\W longifio,;;us· gave values of 28% that of ;B-carotene and, in the same experiment, pro- ~-earotene had e.n activity of 44%. A similar invest- igation was made with (S'-carotene and neo• ~-carotene P which were isolated from Pyraeantha. (62), and wi.th mixed neo• ~ -carotenes prepared by re- fluxing solutions of ~-carotene . The activities found (Table VI) were all lower than that of all•trans- ~-carotene, while the potency of all•SAA! ~ -carotene itself was close (26% versus 28%) to that reported earlier. Since, of all pigments studied, pro• ~ -carotene was the only one whose activity seems to exceed that of all-trans- ~-carotene, it was desirable to clarity some doubts about the relation between pro• ~ •carotene and the all•tran~- 'ZS-carotene as used in earlier tests. All•:a:a.ns- ~ -carotene isolated from ~limulus agreed in most properties With other authentic samples of -carotene. However, there was a great variation in the reported melting points (131 to 178°) and all efforts to raise the melting point above 150° failed (74). Most samples gave values ranging from 1.31 to 150°. The same behavior was observed with samples of "6' -carotene isolated from several different sources (62, 74). Since it is conceivable that the pre,- ¥-carotene and the all•;tra.ns• ~--carotene used in the biological tests belonged to two different stereoisomeric sets, (because of slight, still unrevealed structural differences), it was decided to prepe.re an all-trans- \' -carotene sample from pro• two eubstaru:•s• W.ttle quantitat1v• .information had b9e~ available conc•ning the . . . ' . utillaation ot earotenoids in the chick. • T. K. With (48) carriri ou~ ~he first extensive studies on the provitandn .l er.feet of carotenoid pi,gmenta in the chick. He concluded that crypto:xantbin is a more PQtent prorttaldA A fo.~ the chick than ,,8 •carotene. Some df>ubt exists, however, ot the validity or this statement, since Ii.th did not Qe pure 01'7Ptoxanthin, but based his results on teeding yellow corn, etc. Recently; Ba'UDlaJm and his group (22) reported feeding experiments which sho,red that pun crypto~thin is even inferior in potency to (J•earotene. Oonsidering the importanee of the question; whether; in p:rincipl•, a pol.r-sil carotenQid may have a biopotency as great as its all•WtftP@ .. • form; comparative bioassays were also carried out in the chick as well as the rat (Table VII)• Table Vll B.el.a\ive Pt-ovitamin A Activities of AU• \rw,11,; .i.carotQe and Pro,,i •carotene Subs~ce • I.I l . , 411•:ta,nt• /J •caroten• _ 100 100 , u1..w:111• ~ -carotene (pre}),U'ed trom pro- ~-carotene) 42 42 m-a:ans• ~-carotene 47 (floom tomato paste) h()oio 'l\ •ca:rotene (.f:r-om fxn.cantbf) SJ. 41 As shown by' Table VII, the values for all-trans• ~-;.carotene are considerably h!ghe:r than those (28, 26j) reported previously- (7, this thesis)• but the potencies in the chick and ra.t are identical. The values for pro- 1-S-earotene vers~ al.l•tranp• 25-ea.rotene (ex pro• t •carotene) a.re the same in the rat but pro- ~-carotene has the higher poteney in the chick. This is the first clear case reported where a ~ isomer has equivalent or higher provi·tamin A activity than the all• tra.J!§ form. -41II. PHrl'OFLl,JENOL, A NEW, NATURALLY OCCURRING POLYENE A. IN1'R0DU0TI0N The resolution of extracts from a wide variety of plant materials has demonstrated the presence of a colorless hydrocarbon termed phytofluene .(67,70,71,55,2), end its formula o4ofi64 (! 2H), together with other experimental evidence (72~ plac~ it among the c40-polye:nes with a ca.rotenoid..like structure. Phytofluene is colorless, containing seven double bonds of which only five are believed to be conjugated, but shows a strong, greenish-gray fluorescence in ultraviolet light. Since phytonuene is more highly hydrogenated than the common c40- carotenoids it may represent an intermediate stage in the biosynthesis of these pigments (!55,2), since it occurs regularly in plant organs which are rich in carotenoids. The ratio of pigment to phytofluene, in the materials first studied, was always greater than unity. Some representative figures of the ratio of total carotenoids to phytonuene are: 100: l in carrots; 15: l in ripe tomatoes; and 10 sl in the ripe berries 0£ ~:,ia;acantha angustifol~§ Schneid. An investigation of the blue, anthocyan•containing blossoms of the Brazilian tree, Jacaranda ovalif'olia a. Br. (grown in California) re- vealed a so far unique case in which phytotluene was the major constituent of the polyene mixture. The total amounts extracted from 1 kg. of fresh blossoms were o.6 mg. of phytofluene and o.06 mg. or carotenoid pigments (almost exclusively 1-, -carotene). B. euuACTERlZM'XOI or PllffQfl,UENOl, Since moat oft.he oommoa oarotenoid .hydrocarbons occv ·in nature alol)g with 'l'f:Ll71ng amounts of theu- oxygenated derivatives, it would 'N expected that oxygen•c011t~ining, eolorless polyenes might alse be found. In working with commercial tomato paste, a new, colorl«iis.s polyene was isolated whose prop~rties indicate that it is an oxygenated phytotluene. Its amount is roughly 2% that of the phytofluene content and thu only a .traction ot a per cent of the total polyenes present. Although phytof'luenol has not yet been isolated in analytically pure state, some of its properties indicate the presence o:t oxygen in its ulecule. When partitioned between hexane and SJ% aqueous ethanol, the ratio; concentration in the upper phase& concentration in the lower phase; was found to be 6 a l for pbytot'lueno1 1 whereae the corresponding value for phytotluene is 100 s o. In the partition between hexane and 95i methanol, the ratios were; 2.2 s 1 for phytotluenol, and 100 1 l for phytofluene. The adsorption beha'V'ior is also what one would expect for an oxygen• ated phytonuene (Table VIII). The difference between the adsorption affinities of phytofluene and phytotl.uenol is ~early the same as that between p•carotene, c4off;6, and cryptoxanthin~ C4off 55~oH. This indicates a diftereace of one h1drOXTl group, as do the partition coefficients. As the name suggests, phytofluenol has the same qualitative spectral characteristics as pbytotluene (Figure 15}. The wave length foaitions or the maxima and tninima as well as their relative heights agree v•ry well with those of Ph1tof1uene. The fluorescence of phytotluenol cannot be differentiated from that of phytotluene by the inspection ot solutions or E 10 ·5 320 340 380mp l'igur• lS. lx\inetion curve ot pbytonuenol 1n h~• $& ta.ten 1n the Beekman photoelect.rie spectrophotome;te, (B la relative extinction). • •• • • chromatographic columns. Table VllI Adsorption Sequence or Some Carotenoids and Phytofiuenol on Lime Developed with 15i acetone in petroleum ettier Develope<;i w1 th l to 3% acetone in petroleum ether Lycopene 15'-earotene Neolycopene A /J •Carotene Pbytotluenol o( -Carotene Cryptoxanthin Phyto.tluene is' -Carotene Neo- of -Carotenes Phytofiuenol gives a bluish-green coloration on acid earths (tiltrol) which is characteristic ot phytofiuene and many other polyenie compounds (13,36,72). Phytonuenol could not be observed in extracts of 11 Tangerine11 tomatoes OJ," in a sample or fresh tomatoes (variety unknown). Since the commercial tomato paste used for this wo~k 1a prepared from different amounts of two varieties of tomatoes ("Pearson" and 11San Marzano"), it is possible that phytotluenol occurs only in one of the two varieties. IIJ. STEREOISOIERIC DIPHENltaUTA.DIENES A. 1. §trtrtoisomtrtlm INTR-ODUCTION a PiRbewlBP11en9s In contraat to the earotenoids, where many exampltJs of stable .ll,!· tran9 ~somers are known, there is little evidence for the existenoe of stereolsome):'s in the higher members of the diphenylpofyenea C(,15•(CB:CB)n•C6Bs•* .·. lubn (.31) has expressed the opinion that thia ts due to a lack ot sld.table preparative methods. Ebel (12} bel1e'Vo that a rapid rate or rearrangement would explain why ma117 pretioted stere.o • i ~ s are not obs.-ved. This latter statement is :1n accordance with obsf4"Vations made by Zechmeister and LeRosen ($7,58) in a st.ud7 ot d:lpbenyloetatetraene; where; besides the stable all•kfN! f'orms; several labil.e steno forms were observed on the Tswett column. Methods tor the synthesis or higher diphenylpolyenes, largely developed by Kulm and Winterstein (32), gave only one steric torm, even when the srnthesis was carried out by several different methods. The synthetic products wer• shown to have the all•traps configuration in the cases of .diphenyl• hex$tl'i4tne and dipb.myloctatetraene, 'by means ot l•rq dU'fJ."action stud:tflS carried out by Hengst•nberg and Kwm (19,20}. lubn and linterat-ein (32) deftloped mathemat1e,a l foraulas tor cal.cul.a• ting the nwatar ot possible stereoiso•ers; 1 1 of the symmetrieal 1 unbranched diphen7lpolyenes oontaining n .aliphatic double bonds• · * 1n all further discussion the name dipherqlpolyene is understood to refer to the class or compounds with this general formula. ~-1 When n is even N: 2n-l, 2 n odd N: z1"'1 ♦ 2-z- and for n•l Expressed in slightly different form, these are the formulas used in calculating the number of sterically unhindered isomers in the ease of carotenoids. Although the above formulas are derived on the assumption that all or the aliphatic double bonds can undergo trans~ s!i shifts, Zechmeister and LeRoeen (57,58} pointed out that such shifts would not be equally probable for all the double bonds. In particular, if the double bonds adjacent to the benzene rings were in them configuration, the hydrogen atoms of the benzene rings in the ortho positions to the side chain would be spatially hindered by certain hydrogen atoms of the aliphatic con• jugated system. The same authors predicted, on this basis, that when an all•trans-dipheeylpoly-ene stereoisomerizes, those isomers would 'be favored which have a ~Ms eohfiguration at both terminal double bonds of the aliphatic system. 2. ~ arid gg StUbenes Two spatial f0l;"ID$ of stilbene were first observed in 1897, when Otto and Stoffel (42) obtained the ~-isomel'. £1!.•stilbene ("isostilbene") has been prepared ehetnically by reduction of diphenylaeetylene, C6H 5•C!C•06H;, and by deearboxylation of o<-phenyleinnamie acid, C6ff5•CB:O(C61{5)•GOOH. A tfWJS --->SJl rearrangement can also be brought about by irradiation with ultraviolet light. The light absorption curves of m and tranm•stilbenes have been studied by Smakula and Wassermann (46}, and the differences between t he extinction curves of these two stereo• isomers have been interpreted by Lewis and Calvin (.34). The latttr authors assumed that, in S!l,•stilbentt, the steric com.f'liot between the two ortho hydrogen atoms ot the benzene ring f'orced the molecule out ot coplanai'ity and diminished the resonance. The energy differ.enc♦ betweea s&i and :t:rrrnn•stilbenes (10 kcal. per mole), as well as the. different light absorption ot the two sterie forms, can be reasonably explained by' - the dimini$hed resonance ot the cis form. 1. lMlis_. IPDttitetJu, ~ IM:eh,w-lbutacU.ftAt ?he ta eJtpeQ~ s .~ome:rs of diphenylbutadiene were prepared by Straus (47) as ~ly Q 1905._ 'l'~e 9!i•cis isomer was obtained by reduction or d.iphen.yldiacetylene, and .the cis~trans.form bya r~duc~ion of trps•l,4• dipbenylbuten!,Jle, Q6ff5•o;c •Gir-OJI •C(;1t5. ~J:YS•trag,..diphenylbutadien• oould be obt~ined .troa -. _ e ith~ _of the othe-r stereoisomers by i,rradiation or bf some reduction prooess. Straus obtained the ,sa. . ci,.1 torm as erystals and the·-.d!•:satua 1eora•r aa an oil. .. ( l~l~ and SOh11&l'tz (26), also Ott and Sob:roter 41) Wfl'e al:,J.e to confirm tn. work ot Straus llllirlg ether preparatb·e methods. Zechmeister and Sandoval. (73) madt a. etud7 or some spectral changes which occurred whe:n f!olutions or l£M@•tP1?4J"'dipheny:lbutad1ene were irradiated with artificial light or "insolated" (exposure to sunlight). These authors were able to isolate, by chromatographic methods, a stereochemically hom.ogeneous ,w 1s..-. The quanti:1.tati.ve extinction ourvts w.-e determined foJ! _the . ;k1PQ•xJ:W compound and tor _the Q.I isomer, as well as their behavior toward. heat and iodine catalysis., 2. j iiP!UPS:itk,t -· ~ .$t_l!:t! .St,;;,SU9!A!J:!S d,s/t•ll!Rhen;yl.Ji?Ytadi&ef Since the .~sio?). of the m~thods used in the fie.ld of the carotenoids . ' ,- · should reasonably lftad to a closer correl;ation between the synthetic and nattU"al .polten•, the eal"lier s,nthssia or ihe. three stereoisomer:lc dipnen7lbutad1•nes by Straus was repeated. HQwev.-, modem meth<>da ot chromatopaphy e.nd spectrophotometry were used to isolate and differentiate the thrc,e possible stereoisomeric forms. The SJ'tlthesis of ~s!l•diphenylbutadiene was carried out by the orig1ilal .Straus procedure but also b)" a partial catal,tic !vdrogenat1on of diph~ldiacet,7lene, c6a5•0.C•C:0•C6Hs, uaing a palladium-barium ault9:-te oatal.Jet. ·fJle cis•trans isomer was prepared . in the manner. desert~ b,y s t,raus (47), and the product purified by- cbromatography• .ihe method of inad1at:1on us•d ·by Zeohme~ter an~ Sandoval (7.3) wu also applied to~ the prepara\lon or th• s.u,•tranq form, and it seems to be pr.,ferable to the reduction method used by Strau. Spectr;J. Chara9teristicg ~ lh! Thret St:tr1oisour1 - The profound inf'luenee of the stei-eoohemioal configuration on .the spectral properties of a polyene is illustrated by the extinction curves of the tbree steric forms (Figures 16, 17). The very large differences in the character of light absorption far the diphenylbutadienes are probably due to the fa.ct that both the cig• tr4R1 and the si!•.9ii isomers are of the spatially hinder•d type. While in the case of lycopen.e, a single t1:ans~pi§ rotation shifts the position of the main maximum 7.5 mp. toward shorter wave lengthi,, (in hexane, measured :in the Beckman spectrophotometer), one ~1~.GI shift in th• diphenylbutadie~s ca~ee a wave length decrease or · the main maximUDl of 14•15 mp. (Table lX), which is in good accordance with values repo.r ted tor stilbene, where the corresponding decreal!le in the wave length position (in ethanol) is lS mµ {46). The data gi••n 1n Table lX constitutes additional. evidence which supports the assignment ot configurations by Straus (47). The lack of fine structure :in the run~ntal ·~ or the two s!i isomers ean be correlated with similar observations made in the study of poly•~•lycopenes (page 28) • - - " r ' ~ 5 / I I I r>J9,-, I t ' 4 I I I 'i I I jv I ! 0 I X I . I w- 10 ~ .f= 0 3 I I i ,Jt--- ~ --\ 1I \-- \ If/\ I t' 1/ I .,, '";\. ~'J_ I ] I y 2 \ I \ ~~~ \- \L_~ I \ - _\_-/ v1' r-- '- I ~ '~ \ -,~ ' - \- ,~~ ~\ / _ , '-\:~/ \ - -· __, I --=-i.,, \ - \ _' \--j' ~ I I ""- 250 300 ~ 350mµ. lol.olllu e:x\iD.Q\ion cunts ot the· three ateNoteo•no UJh-.l• bu~l.-.1 Sn haane1 ..--, klaf•)llu ~ J ~ • ..,.. •• - - - - OQIIIJ)OunclJ •••• , .!U"'al. ooatp0UD4J --.•......~ -. .,__ t.u.ne oettal181- ot ADJ' of the foregoing atlutt••• • I (, I ,-'-\ I i \ \ 4 PJ I '~ I I I \ I \ I \ - I I 3 1J \ I q'" I 0 ,___, - .__ )( 1....: E :Oeu I I I :w ~I - - I I 2 ~-1 I ~; I i I I ~ I I I I I I I \- I - ~, - I ' \- : ~ V I I_ \_ \ I 300 I \ 350mµ 1 ~ :&.7~ Jtol.-ular ext.action curve of SY·W""ciiph•n,lbUtadi•n• 1n ben:atn•• ...--1 fr•sh sQlut1e>nJ ca~.7au. ... .......,_ •• aftfJr iotitn• • the ilOdela •ot the three 41ph-,ibuW:lenes giYeJ;l 1n li,gu:r• 18 .show that the E>verall .ah•~ ot •-the J!k~it!N ,molecule ·.is (\Ut.J ~t~•nt tr• that or tbe H'IPf•U:AY cw the m •!!,. form. The open-4 V•ahap.- ot .the 11011..,GI 1•om.er should giv• rise to a characteristic iibs9rption band in the •~ct-rum, as is obsened in the case of a carotene>id stereoisomer with a s i ~ oontigu:ration. Although a distincti $ • peak does not appev 1n the speOtl"al CUl"Ve of sJA•ttanf•diphenylbutadiene it has th♦ highest extinotion ot the three steric forms in the region bet.ween 230and24011}1, (ot. Jigure 16). This wave length region is approximately where a !.14• . . \ • peak .would be expeoted. In the cue ot the 040 carotenoida, \be a.length dittereno.e between the poaitions ot the longest ••• length ~ and the tJl•p..t 1• about 142 mp (in heme) but th• ooneapond.llg tlguz-• tor Jdl•$rJmt•c:l.pheD7lbutadiene is onl;r about 105 aµ. In n• ot the tact that the resonating ayata is much ehorter in the latter case, tti. value of 105 Jl}l ia reasonable. Th• deci-e.ase in the inten11t,7 ot the fundamental band, as meas--4 by the area ben•ath the absOJ."ption curve, closely par.Uel• the deol"eae• in inteiutit7 which one finds in polf"!'Sil•l~openes (pap 27). Qhre,tor,t1:PM1 S19ueu91 • The ohromatogra.J)h1C sequen¢e ot ·t ;q t_.e etereoiaomer1o diphentlbutadients observed on an alumua column, ulng petroleua ether as a deTeloper, is, trom top to bottomJ !n-Pn-ktPI, a&I-WMI~ and SH•SA• Thia decrease :tn adsorption aff'init1•• with increasug nwa'b4;r ot m double bonds 111 characteric tor th• 1700,ene .., and tor mosi s,u.-iaom.-s in the oth•r caro~oid bydrooarbon ae\a. lboDdtD!• QbAracteriptiog • fhe dit'terenoo in th$ nuoreaoeno ot the tlu-ee swtca, torma ot diphenrlbutadiene are ot great help m \he 1,4 Oiphenylbutadienes --- -- • - - ---- - -- .- -- . --.. cis-trans cis-cis Figure 18. Models of tips•t1;an1• (top), ~•H:f¼ng• (bottom, left), and .. cie•w_•diphenylbutadiene (bottom, right} .. (Values: c=c, 1.33 ,:, C•C, 1.46 A.; and C:c-C angle 121i,O 20'• The dotted lines conn,ot the ends of each resonating system). lo~ation of zones on the chromatogram. The zone of the kana•ttw form is easily iderit.ified by its intense bluish fluorescence in ultraviolet · light. The ois•:terans and m-ci§ forms do not f'luoresce either on the 'l'swett column., or in solution., but on alumina their zones can be located easily, since they queneh the weak fluorescence of commercial alumina. Zones of all ·l;l>.:ree stereoisomers on the chromatographic column also may be located by brushing the extruded. ~olumn with permanganate solution (61). The liquid chromatogram method (U$ing alumina and petroleum ether) . ' ' 7 proved to be ·llBef'ul for a sharp separation of the gis-trana and cig•s.Y! isomers. fhe filtrate from the column was collected in. suitable fractions &--id a rew drops of each fraction were t,sted by adding. . a drop of dUute . . ,, ~ iodine solution (in petroleum ether) a.n\i illuminating £-07 81 few minutes. I£ either of the .$ii forms is present; •,an inspectio-n with ultraviolet 'light will then reveal fluorescence caU!iied by the catalytic ': formation of ;xrans•j,rans•diphenylb-11tadiene. Thi• test is sensitive 81\~ugh to p.-m!t tbe detection of one to two micrograms('! .j,.n a two...ml. sample. ,. • ' AW Sta.bilit;v • The stability ~f'\~he three forms ·t;o hea\t was tested . I in the £ollowing ways. \'.' In petroleum et~r solution, all thr4 oompoµnds are stable tor at least two months whJ\ stored at 4o. When; h~ne \ . • 'i \, solutions (b.p. 67°) are refluxed inan ~-glass apparatus~ da\:kness h • ·'I, \ ~·- for rorty--five minutes, all three forms arij uneha..~ged, • . • ·1 1 ~\ ; • ln fil.notbet '• et or ,:- : '\ \:.3 experiments., samples of each steric f~t'm wet;e sealed in evacuated PYl"•l :1 ; .;, tubes and kept at 205° for ten minutes , in d~kness. : ,_; • ·,. '.~ The tra,ps•t;:ang ui~ ' ,• !: \·\ •\ .91£•:S:W:P:! compounds were practically up.~.feeted and the .£!l•~ to.rm showed. less th.an 5% ·isomerizntion. fhoto:§tab!lity • The 'behavior or tlie three stereoisomers toward ~~gh~ !, ., \ was tested in two different types of ~xperiments, a) illumination of hexane solutions (20 mg. per liter) in pyrex flasks with a 250 watt light bulb for several hours, and b) exposure of more dilute hexane solutions (3-4 mg. per liter) to intense sunshine, in transparent quartz tubes, foiQne to ten minutes. In the a) series the ci§-trans•isomer remained praet:l.c~lly unchanged, even after seventeen hours of illumination, at which time, the sj&•cis and :t,ra.ns•t;ranm isomers (Figures 19, 20) had be.en almost(luantitatively converted to the eis•tra:ns isomer. The conversion of the trans-trans isomer was nearly complete after two hours illumination; while the ~is•cis form required thirteen to fourteen hours under t he same conditions. No significant photochemical destruction of any of the three isomers occurred during a seventeen hour period of illumination. In series b) the trans•trans isomer was l argely converted to the •,n;ft;e--f i::ts~ •. eis-trans form by a one minute insolation, and almost completely after ten minutes exposure (Figure 21). The cis-gis isomer was almost completely converted ·to the gis•trans configuration after ten minutes isolation (Figure 22), while the cis .. trans .form was practically unchanged under these conditions. There was 8-10% photo•destruotion in these experiment$• In both series the extent of irreversible changes was estimated by carrying out a.n iodine catalysis after the completion 0£ the experiment, and calculating the final concentration based on known equilibrium values of the stereoisomeric mixture obtained by catalysis of any ot the three steric forms (Table XII). This mixture consists of about 9"11, trans•~ and .3% ois•trans form, while no _g;\s-~ form has been observed so far under these conditions. The necessity for light in effecting iodine catalysi s of carotenoids -56- _ ,, !-, 5 j~I I - I ~4 I \ ~ \ I I \ f c;t ~'\ ·~ ;1//~~,{~ 1/ ~' 3 I 0 X ~I _;E Ou / I EW ~: t ~ t - f/'.J , ~A , ,' 250 - \ \.,. :,- \1 \ \ -~ - I \ \ ~\' -~1-I \~\ \ - ~- - \\\I _ I \\~ \ ~/ I I ,_ \ \\ I ' 'i 1/j,' /2 ~~-\~ "~ ' /I/" ' - ,~ 1///, y;1/;Jxi ..,JS ~ ,~\\ -~. \ \ \ / 1/ I •f 2 \ I - , -- 300 350 mµ Figure 19. Molecular extinction curve of S!i!•ois•diph~nylbutadiene in hexane, and its shift toward the curve of the eis•ti-ans form during illumination with a 2;0..wa.tt bulbi - , fresh solution - - - , after fortY""five minutes; _ ... _ - , th;fee ho~a and forty•five minutes; and ~ , seventeen hours of illumination - - - -, after iodine e.a talysis at the end ot sevente,en ho'Ul"s ot il.luniintrtion. t-----t----t-+--+------+--+----+-----+-_____j.__J___(' --t-----+---------1---1 5 i--------+--+---+-----+--------l---------l--------1----___j___L__l__ r·r, ' ----+---+---~ .I\ if ------t------f------1---1: ----t----j---+---~I I '/'-J~ t--+---+----+--+------1---------1---~--L--- rv 7 4~-+---+--+-+--+----+--+----t-~ I t---t---+-+------+------jl-------t-----------l--___j__ ~~ ~ \~~ f \\,~ 0, v I 0 X --+----+-----_,________, I ~~\ \ ._/"\ +----+----I ~, ."\ 3 t---+----+-+----+---+-+------l---- ./_ ~\~, \ o, -·e= t----+---+- Oo IiI +----+----+-+---+-- . ~- w \ \ • '\, . I I - - I - - - - - + - - 0\ \ ~\' o, \ -+------1------1-- \ . -----+------l--___,_______,_ A \\ \\, , ~j/ ~ \ •\\---+---+-,,.II ~ -~~-\ \---~ \,' ~ , / / \ ' Figure 20. 't : -+-----+----+----+---l----l--------+\ I 260 --+-~ t ~ I 230 \ // /J ~~-~~f I----+--- - + - ----+-----< \ \\ i~ .___--l--___,_~,,-,'l'i----l- I \I ,:/ ,________,_____J_ \ ------+-------- 1 \\ « // ----+------f l I I \\ 2 1-------+---+------+------+---+---l---1 --:"/~~~, \\ ----+-------f - I 290 320 350 TTIJ.l. ~ol.eoul&l" extinction curve of ;s:anp-trg:,p-diphenylbutadiene b hexane, and its shift toward the curve of the .ii!•SIDI ~ during illumination with a 250-.watt bulb1 - , &eali 1olution of the trans-trans eompoundJ _ - _, after fi£t,ea ld.n1lte11 -•-•....., fort;r•tive minutee; ••-, one hour and. tort,-ti"i'e 11W1U\e8 - o - o.- , eight ho~s Ulumina\1.onJ -•--•...,-, alt.- iodine catal)'s1s at the end ot seventeen noun illumlna\lOJ>.. --58' • 5 N/\ l I \ -\ I ,......._/--\ I \ 4 I I I I I \V 't / I r, I I ,' _ \ I I.,--,, I ,,, _ I I - I / ~/; 'I I ,'f 1/'/ J' I 2 I ... ~ ,..,,'"'-.: '1/ \ \ _ '\ ' ' - ,1 '-\ \ I \ \ \ I \ \ \ - \ I - '. \ \\ .\ \ \ \ \ ,\ ~ , \ - ~ I, . \,_...7.,""' .,, ~ \ - \ \\\_1·\ \ __ \ \, - ' ,l \ \ ,\ I I \~ ~ \\,, - 1/ 11 - - ,J ,.Ii/ ,., ' l ,'I •, I \ \ \ ,,// II ,, ""°' \ ' 1 ,1 ~ ~\ \\ \ , I ,, ~-- ~ , _' ' - , - - \ I '\\' -.. ~ - ~ I 250 300 35Qmµ figure a . Molecular extinction curve of tffiWS"":sJUl8•diphen7lbutadiene in hexanet and its shift toward the curve of the .9.!&• tJ:an1 form during exposure to sunshines - , fresh solution; - - - - , after one minute; • ··•• ,,ten minutes of insolation; - •...,.. •, after iodine eatal;ysis at the end o£ ten minutes ot insolation. l',I I \ I ' I \ I \ , ,...y I 4 I ' \ I ' \ /: I I I I , -/1 - ~~ '\ I I I, 1' ~ I /, ~Ii I - -I~· I II I I e-/_ 1 l/ ~'\ 1 - f- \. \ -~,Ii' \ C?<-+\\ \ ,_._I / / ~ , - \ w ) _/ ~ II;' \. / \ \ 1=-~, I .,,,~ r" ' \ I ~ \ \'- \ I ,- \ I \ I- \ \I \ - \- \_ \ \ ~•f- ' ~ \_ I ,~\ /-, ' { 1 ,/ ,--1/ '-' I - \\_ \' \tj 350mµ 250 11gur• 22. Mol•culer extinction ourv• of s!l,•ej9•diphenylbutadi,ne in h.-..ne., and its (:!hi.ft toward the curve of the 0~1•\TMQ ton dlU"il'lg exposure to sunshines - , fr•h solution; .,,.,.,..:-1 an.-. ten minuws ot insolation; - ~- • , after iodine ~•ta1Js1,s at t~•· ~d of ten minutes of ineolation. • -60- is well known {49). The s ame phenomenon takes place in the diphen;rl- butadiene set, as is illustrated in Figure 2.3. In darkness, after the addition of iodine to a hexane solution of ~•eis diphenylbutadiene, there was no observed change within half' an hour. Exposure of this solution to light caused a rapid conversion. Some idea of the mechanism ot this rearrangement was obtained by taking the complete extinction curves after definite times of illumination. figure 23 shows that the process, Ji!•s!l--+-S1•tx:w~tr§J.!§•tray proceeds in a stepwise fashion. The appearance of any significant amount or the trans-traps isomer in tho early stages or this process would be manifested by the appearance ot bulges o:r peaks at those wave lengths where the maxima ot the trans•:ttsn@ form are located. l!tsteil 91. Stability; at, .ill!. Solvyt-free Stereoisomgrs • Although .QH•;s:es•diphenylbutadiene appears to be the most stable of the three steric forms when illuminated in dilute solut;ton, it is the least stable in the solvent-tree condition. The trpa-trans and S!§•cis forms crystal• Uze easil71 but attempts to OJ"ystallize the .sa!•trans compound failed and y.tel.ded an oil, which, on exposure to light, deposited crystals of the kMl•WIJll isomer. The arrangement of molecules of cts•trang-diphenyl• butadlene into a crystal grating might be much more difficult than for the Jth~io members ot this set, considering their bent shape, and could accout tor the failure to obtain crystal,a. . - - -- - f\ -61- 5 I 1-1 I/ I ,0- \ / I I t' \ I I 4 / 1/-\ !/ - \ '\ I I I I ' I II °'-":_\~ \-r- I ,1 7, ~ I ~~ I \ + - Yo\-~ ~ -I 0 X . ~ll -=E Ou Ir E- t1,i ;,:~, W 2 ~I K I f-\ ~t\ I \ /f,f I "- '' \ ~... , -\\ \_ ~-'\~' ' 'f ,1\- '" ~~ I ~, \\ L- /~½z1r I 1 0 I !' \ l;j- -'q',q. ,-~ \ I ~3 \ '0, I ! ' Ii I I II \ \ Y.\'\-- '~~ ,~~ (/ 1/?I!/ I j ~\~\\ I~~·~ . . ._/-- \.+~~ 0 \t' t1/// ' ~ I 250 ~,~·N - 300 350mµ Figur• 23 . llolecul.a:r .xtinction curves in hexane; influence ot illumination on the stereoisomel"ization of a,U•.Q!-d1phenyl butad1ene caused by tod1.n.e ca.talysi.81 - , fresh solution with iodine, kept in <larkne:ss tor: thirty minutes; .... ..... , · after eighty seoonds; - ""' - - • one hundred and forty seconds; -x~-, 200 seconds; - - - - - , 320 seeoade; and - - - ... , •2S minutes illumination. Aq)Snowleggent - This work was carried. out under the guidance or Professor L. Zechmeister and has been published in part, (5,18,62,6.3, 64,80). I wish to thank Mr . Borge W ill$ and Dr. A. Sandoval for their co.-operat1on in many phases of the work. Professor H.J. Deuel Jr. and his group carried out the bio.assays for provitamin A activities. I am indebted to Professor F. W. l ent and Prof'essor E. G. Anderson tor supplying experimental material, and to Professor A. J. Haagen-Smit, the late Dr. G. Oppenheimer and Mr. Q. Swinehart £or microanalyseo. Mr. Kenneth Koe and Mr. Lawrence Wall.cave gave technical assistanee. I. lSOLA'l'I0N OF SOME -eis GAROTEN0IDS · • A. INTlWDU0TI0N In all or the experimental work reported in this Thesis, certain standard techniques, equipment, and reagents were used. In order to avoid a useless repetition of detail, a description is now given which applies to all further work, unless specif'ic mention is necessary for a partieular case. 4dpor91n\p • Two brands of calcium hydroxide were usedJ Shell Brand, CheJi11cal Hydrate, 98% through 32!> mesh, W estvaco Chlorine Products, Newark, Calif. (no longer available) and Sierra Hydrated Lime, Superfine, Unite(l States Lime Froducts Corp., Los Angeles, Calif. It was sometimes necessary to mix the limes with Celite, #545, Johns-Manville Oo., in order to obtain a bett6r rate of filtration. The alumina used was Activated Alumina, Grade F, 80-200 mesh, Aluminum Ore Co., East St. Louis, ru. The alumina was re-ground to -200 mesh, and lllixed with eelite. The "sllicic acid" was Merck reagent silicie acid plus 2~ aelite. §olyeqtg - Petroleum ether was used in most chromatogr{:lphic experiments. The brand was Skelly-solve B, boiling range 60°..70°. Benzene used for chromatographic work was technical grade, and that used tor spectra and crystallization was reagent grade. Acetone and ethanol refer to c.p. acetone and to 95% ethanol. A commercial grade of methanol was . used. Hexane used for spectral work was prepared by treating Phillips commercial grade hexane, or Eastman practical grade hexane repeatedly with fuming sulfuric a.cid until its optical density. was near that ot distilled water. Chromatoqaphig QgJ..umns. - Unless otherwile stated, the term •column11 refers to a ,30 x 8 cm. cylindrical column. IJ?!C'k!J. l4gasurements - Visual spectra were taken on an Evaluating Grating Spectroscope, Zeiss, light filter BG-7. Quantitative extinction curves were taken, using a Beckman photoelectric spectrophotometer (4). Measurements were customarily made by using average values based on two independent weighings. Oenerru; JOH@ - Evaporations and concentrations .were performed in yacug (water aspirators) while a stream of nitrogen or carbon dioxide bubbled through the solution. Sintered glass tunnels were used tor elutions, and in general, where possible, all-glass apparatus was used for evaporations, washing etc. Melting points were taken on an electrically heated Berl block in capillary tubes. The capillary tubes were either sealed under vacuum or .filled with carbon dioxide and then sealed. IodiY.,~ C§tal.Ysis - Iodine (1$ of the pigment weight) was added to the solution in volumetric flasks and fifteen minutes or illumination followed, using two 3500° Mazda. white fluorescent lights (40 watt, length of the tubes 120 om.; from a 60 cm. distance). B. OAROTENOIDS FROM:PJMGANTB& fixttact1oe - lresh PYracantha. bei-ries, picked. in Southerm. Dalifornia, 1n December and January, were stored under methanol in darkness at 4° until they w,re worked up. It was found that, under these conditions, even half a year of storage would not essentially alter the results. Sixteen kilogr~s of this material (fresh weight) was drained or methanol and the JllOist berries were coarsely out in an electri c chopper. The material, e.ftel" ~Ying be• kept un4er f:resh methanol for twenty-four hours, was centritugeci' and the greenish•brown liquid discarded. The exti'action was catried out with 1.5-kg. portions by shaking mechanically for fifteen minllteswith l .l iter methanol ♦ l liter petroleum ether; which formed tw() ,.liquid phases. The residue was allowed to dry on trays tor five-s:lx hours $bd then ground 1n a Wiley mill No. 1 until the particles passed a 1.5 --• sieve. The extraction was repeated three more times, as described; and all extracts were, combined. The polyenes contained in th• 11ethanoi layei- were transferred 'by- cautious addition of water (in 20-liter separatory funnels) .into the petroleum ether (30 liters) which was then washed methanol-tree. The dark red solution was dried over anbydr9us ealeiu.m chloride and concentrated to 1.5 liters. When kept at 4° overnight, a greenish gummy material appeared whieh was removed by suckirlg the solution through a 4•cm. layer of calcium carbonate on a sintered glass tunnel. 'fhe adsorbent was washed with a small volume of petrol•um ether and the latter added to the main solution. Isgla)ion - The petroleum ether solutien just mentioned was developed with the same solvent on twenty-one columns of Shell lime. Each column showed the following sequence after half an hour of development. ' (Figures on the lett denote width or zones in mm.J some very narrow, pale interzones are omitted. 2 brown 2 red 5 orange 17 red-orange, lycopane 40 pale orange, undifferentiated 17 red-orange 40 orange, prolycopene 18 yellow 6 pale in.terzone 36 . orange, p'.ro- ~-carotene 7 yellow 2 orange 65 orange, l3•oarotene 10 yellow 8 fluorescent; partly overlapping preceding zone The development waa continued until nine-tenths of the zone had been washed into the filtrat•• -carotene (This filtrate contained considerable amounts or phytotluene). Each column then showed the foUoring sequence 3 brown 6 orange 7 red•orange, lyeopene Section I 40 pale (!)range•,, un.difterentia.ted 23 nd-o:tange Section II 48 orange• prolycopene 20 veey pale yellow 42 orange, pro- ;s' -carotene • Section• III S yellow 4 pale orailg• 6 alniost. eolorless Section IV 10 orange,, (3 -carotene After· elution,, the corresponding Sections I,, II, llI,. and 'IV ot· the twenty-one columns were eombined., The above Section I consisted mainly of all•ttoos-lycopene which was e~ti~ted photometrically~" 1. Pre- ~ •carotene - The petroleum ether solution from Section III va.s rechromatographed on three columns.. The combined main zones Y1ere eluted,. washed free of alcohol and dried.. 'l'he solution was then completely- evaporated and the residue dissolved in a minimum amount of benzene. Pro• ~ •carotene was obtained by adding absolute methanol drQpwis-e at 20-258 , with stirring,. until crystallization started,. cooling in ice water and cautious addition of methanol up to three times the initial volum(ll• After recrystallization at room temperature and washing the sample with::, x 10 ml •. or hot methanol in the centrifuge tube, 211 ·. 0 mg. of pure .pro• ~o •carotene. was obtained, m.p. 134-5. In a miXed chromatogram test there was no separation from a.n authentic sample •. 2. l;roly;copge • The elua:te of Section II (from 21 columns), after transfer into petroleum ether, ws rechroma.tographed on twelve columns. 10 pale orange 35 almost colorless 2 yellow l purple -----------·-------------- ...---- 10 pale orange Section V 2 ;yellow l purpl.e Section VI 4 pale· inte,rzone --·------------~-·--~~~----• • ' ,,· . • I S red•orange l pale interzone Section VII 10 red-orange . 1 pale interzone 6 red-orange ------------·--··-----------·- Section VIII 35 orange, prolyeopene 8 yellow Section IX 3 orange Section VIII was rechl'Qmatographed on three· columns. Upon develop- ment with 4% l.lcetone in petroleum ether, the three main. zones were . ,· .. ' combined and, after transfer into petr0lewa. ether~ the latter was evaporated to dryness. The residue was erystallized as given above tor pro• ~ -carotene; af'ter crystallization, 7.35 mg. of pure prolycopene, m.p. 111.; • 112.5°, was obtained, which did not separate, in a mixed chromatogram test, from an authentic sample. Section VI was developed on three columns with petroleum ether+ 41,, acetone. 2 brown 8 red-orange 4 pale interzone - 80 dark orange, poly-cis-lycopene I 100 several minor zones (added to Section VII) 3. folr•cia-1:vcopenes I - llI 'l'he three .combined 80 mm. zones were reohromatographed on asinglt colWllll. ·ne main zone of this chromatogram was transferred into petroleum ethe, and evaporated. 'lhe crystallization ot its residue was carried out as for pro- ~-carotene (yield, 45 mg. ot poly-~is-lycopene I; after recr;rstalllsation, m.p. 93•95°). The total ot Sections VII (from 12 columns) was developed on twelve columns with petroleum ether ♦ 8% acetone. 8 pale orange 6 purple 4 pale interzone 10 pale orange 6 pale interzone 4 pale orange 10 pale interzone 12 dark orange 6 pale interzone 56 pale orange 18 red-orange 4 yellow Section I. 3 pale interzone -.---------·•--..---..........______ 40 red orang• Section XI 8 pale yellow ---~-~--··---------~---~~-- 40 pale red 16 pale orange ,_ __..,. ....... ___________________ .., Section XII S•ction X (from 12 columns) was developed with 6% acetone in petrole'WD ether on thi-ee columns and gave the following sequence. 15 several minor zones colorless 30 ---------·-•·· -------------..........................___________ _ 80 pal• orange • Section XIII 40 red-orange Section XIV 30 several minor zones . __ _ _ _ _ _ _ . . . _ , _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ ill!" _ _ _ .Section XllI (frca 3 columns) was developed with 6'/, acetone in petroleum ether on a single column. The main zone was transferred into petroleum ether and evaporated. The residue was crystallized from benMDe and methanol. Por the crystal phot.~graph in figure 25, poly- sa•4rcopene II was crystallized from petroleum ether ♦ methanol with addit.i on of enough ethanol to prevent t wo liquid phases. The yield after recrystallization was l.4 mg. of pol;y•,sil•lycopene II, m.p. 85-87°. Section XIV (£>:lom 3 columns) was developed on a single column with petrol•• ether and 6% acetone. The solution from the combined main zone was treated as j1U1t described. Yield, after recrystallization from benaene-Jllethanol, was 19 mg. or poly•a!!•lyoopene III, m.p. 105-106°. 4. AJ 1•pans- "6' -caroten1 Section XI (from 12 columns) was developed on £01.r columns with -71petroleum ether and 4j acetone. The combined main zones were eluted, · transterred into petroleum ether, evaporated and cr,stallized. Att•treeryst~ization, 42 mg. ot all•treo~ ~ -carotene wap obtained with the constant m.p. 128-129°. In the mixed chromatogram test it did not sepuate trom an authentic sample. s. In: 't{ -caroten1 f .Section III (from 12 columns) was developed on three columns with petroleum ether and 6% acetone. The three combined main zones were transferred into petroleum. ether. After a like development on a single column, the solution from the main zone was evaporated and the residue was er7stallized. • At'ter recrystallization, 47 mg. of neo- ~-carotene P, m.p. 89-90° _. resulted. The main pigment obtained by iodine catalysis from this isomer did not separate .from ~-carotene(,!! Mimyl.,y} in the mixed c~omatogram test. 6. ~oly•c1s-lycopenes IV • VI Section IX was developed on three column3 wi t.'l petroleum ether and 2J acetone. S brown 2 eolOl"less 18 several minor zones 100 orange, prolycopene 60 yellow (lower part orange) 8 red-orange, pro• ~ -carotene 10 yellow After ti-ansf'er into petroleum ether, the three combined 60-mm. zones were developed with petroleum ether and 4% acetone on two colUIJlne. -72- 15 several minor ~ones 1.30 bright 7$ll.o-,v · 40 yellow and ~le orange zones 4 red-orange The two combined 40--mm. zones were ·9-eveloped on a. s:i.ngle column (25 x 4.5 cm.) with petrolemn ether and 4'/, acetone. 10 several mino~ zenes 6 colorless 60 pale yellow, ~o:J..y•ois~lycopene IV 5 al.most colori'.es's 80 yellow, pol7•$1j•;rcope~e V 3 pale yellow 20 7ello.-orang~~·f poly1,i!!•lycopene VI Each poly-cia compound was reehr$~.~ographed. Since the quantit7 ot these pigments was not sufficient: ~Ol\the preparation ot crystallin• samples, in order to determine the e~~el1on curves; the solvent was • . :, ; \ displaced from the columns with 65 m11~\: ,r ,i>'t1cally pure hexan•• After (; -r· elution, the respective pigments wer~ .\:~~a~erred into pure hexane. The ·t·;_, . 'I ~;_ :_. , concentrations ware determined; after1i\~ne catalysis, . using the ·\· equilibrium value ~ 0 !;. =14.6 x 1o4 ~:t\ 4~-469 mp. which is the average . \, I • of the figures obtained with four diffel'~nt/ isomers end only slightly . ,: different from some data given earli~ (5~). ' '\' 11gur• 24. Prolyoopene ( er) and Polr.£11•1,copen• I (lo..,), cr19talliaed tro benzene and m thanol. gn1t1caUon 430X. • • • 0 • 0 0 0 " 0 • • 0 ,, 0 • 0 ,1 • 0 Table X Molecular $Xtinetion Coet:ficients . or Some Poly-si4• lyeopenes (!15 fn:aeantm;) and their Stereoisomeric Equilibria at the Maxima (italiciaeq) and Minima; in Hexane. hesh solution After iodine isomerization mo1 •._ mol.• •. mJl EX 10• 4 .• l cm, i1l}l Ex 10 4 1 gm • . Poly-,w.- lycopene I ~ 12. 0 485 9., 3 ~ 12. J 4f>7-8 14.2 458- 9 10. 5 449 9. 9 ldi:,- 5 12.3 ~ 10. 1 375 1.4(4) 361 2. 8(6) .351 2.0(3) JG/. 2. 0(7) .311 o.6(4) .317 o.7(2) ~ 1.4(4) ~ 3. 6(0) 290 1. 2)1) 26; 1.2(0) ~ 2. 7(8) 255-6 1.4(5) 240 1. 9{2) 21+1 1.0(9) m 2. 0(3) - 1. 6{6) 226 -76- Table X Molecular Extinction Ooe£.ficients ot Some Poly•eis.,,. lycopenes (,s fn:aga.n'tha) and their Stereoisom.eric Equilibria at the Maxima (.i~t(d.z~g) and :&1inimat in Hexane • Fresh solution .Atter iodine isomerization mol. mol. m.)i E x. 10·4 l om. E x 10""4 1 cm. Pt?lY•$•lycopene II '2§ 12,0 485 9.3 465-6 10. 6 IJtl. u.. 2 453-4 9.9 449 10. 0 441-2 ll. l ~ 10. 2 375 1.5(5) ~ .3 . 0(2) 351 2.1 (5) ~ 2.2(1) 312 o. s(1) 316 0.7(6) ~ 1.5(1) ~ .3 . 6{9) 288- 90 l •.3(2) 265 1. 2(1) 256 .2.4(4) ~ 1.4(2) 244 2.1(8) ~ 1 . 0(2) m 2. 6(7) 225 1. 5(1) Tabl• I ~ontinued• Molecular Extincti,on Coeff'iciell~. ot Som• Poly•ci•• l.ycopenea (s F;n:acan!hi) pd ~heir Stereo.:1$ollleiic EquUibr1a at the Maxima (italicizes) and Minima• 1n Hexane~ Fresh solution After ·1odine isomeriaatioa it!§ 12.4 485 9.7 AY 10.s A6'Z~i 14.7 4S8•61 10.2 448 10.4 MJ-6 ll . .3 ~ 10.6 375 1.6(7) lgl 3.1(0) .351 2.2(4) JG/. 2.2(8) 312 o.7(9) .316 0.8(2) ~ 1.4(2) 422 3.8(2) 280-2 1.2(0) 266 1.2(6) 2'5-6 2.-5(.2) i,S:§ l.4(8) 240 2.0(7) 242 1.1(0) 11&· 2.S(9) .ill 1.7(1) Table X ...continued• Molecular Extinction Coefficients . of Some Poly-Si!• lycopenes (s Pn:acantha) and their Stereoisomeric Equilibria at the Maxima (italio;\ztd) and Minima, in Hexane .. Fresh solution After iodine isomerization mol. Ex 10·4 l cm. Ex 10• 4 mol..• l cm. Poly•cis•lycopene IV a!IJ. 11.4 485 10.0 w. 14.6 449 11. s ~ 10. 6 442 ll.6 412 8. 9(7) 375 2.4 ~ 9.0(7) l2! 3.4 350 .2.4(6) ~ 2. 5(0) .'.318 1.1 m 4.2 .315 1 . 2(7) Table X --Continued:• Molecular Extinction Coefficients_of Some Poly•si!• lycopenes (s P;n:acantha) and their Stereoisomeric Equilibria at the Mrod.ma (italicized.) and Minima, in Hexane-. After iodine isomerization fresh solut_ion mol,. mol ... EX 10•4 Ex 10 4 l cm. 1 cm. Poly•'Cis•lyeopene V ~ 12.1 485 9.6 46z-a 14.6 449 10.4 431•6 8.9(8) ~ 10.7 416-8 8.0(7) 375 l.8 412-4 8.1{3) 361 3.2(1) 351 2. 3(3) ~ 2.3(7) 317 0.9{8) ~ 4.1 314 1.1{8} Table X ~ontinu•ci- liolecul_. E:rtin~tion, Coe:,tr.iei:ents of Solil♦ P.oly•!.k• l70openea ·(s•fnacii»ha) ·&lld their .Ster~isomeric EqUUlbria at the Maxima •(i,ttfuiQizeq) encl Minimat 1n B~e. • Attt1.' iodine lsomerisation mo1 • . Ex 10•4 1 cm. Poly•ga, lyeopene VI IJl 31,2,.4 8.1(6) 1.1{6) 497.g 11.9 485-6 9.8 467-8 u..6 448 10.7 442...3 10.8 374 1.7(6) ~ ).0(7) .350 2.2(8) ~ 2.3(3) .316-S 0.9(5) ~ 3.8(4) -81- Q. POLY-CitJ•LYCOPENES FROfl THE ''TANGERDUiP TOMATO ·- Ep.raction - 87.6 kg. of ripe tangerine tomatoes were sliced into quarters and ground with methanol in a "Waring Blendor" until a fine mash was obta11'led. Tbismaeb was filtered through a cloth lining 1n a basket centrit\lge. The first liquid was round to be essentially free of carotenoid pigmel'lts and was discarded. The moist residue or tomato pulp was extracted by shaking 5 kg ••port:to.ns with 6 liters ot 1 a 1 mixture of pert.rol•tml ether and methanol (thei-e were two liquid phases) on a mechanical shaker tor 20 minutes. The mixture was filtered through a cloth•lined basket in the <1entrituge and .the filtrate saved. The residue was extracted once more with a mixture of petroleum ethe:r and methanol (2, :L) and filtered as abov•• The residue · (which was nearly colorless) was. discarded. The conibined filtrates were pouioed into 20•11.ter separa~ry funnels and the pigments were transferred to the petroleum ether by cautious addition of water. • the aqueous phase was discarded. The petroleum- ether solution was con:c~ntrated 1n vacuo~ a tot al volume of about 3 liters. This concentrate was filtered through a 6 cm. layer of celite in a sintered glass tunnel to remo•• ~ ma:t;.erial. The eelite was washed with 500 ml. of petroleum et.hfd' and_thewashings combined with the first filtrate. Ig92,t\tism • The petroleum ether solution or the pigments 1'118. chr~to• graphed 011 titte.a columns and developed with 5j acetone in petroleua ether. Th• top to bottom sequence or the pigments on the eolumn• after_the lowest pigment ion• wa-s near the bottom, is given below. left denote width ot zones in mm.). (Figures on the ~-------·------------..·--------...--- 15 pale buff 20 2 red zones Section I S pale orange ----.-------------.--..------------~------ 4 very pale orange 10 orange 10 pale orange Section II 22 orange 10 yellow 3 Grange-red ---·---------------·-·-------------.,-- 4 almQst colorless 52 orange (prolycopene) Section III 20 yellow-orange 30 pale yellow 2 orange 8 yellow•orange Section IV 3 colorless 8 orange ( ,S •carotene) 20 nuoresc•nt zone (phytofluene) tlltrate nuorescent (phytofluene) -. . ------·-·---~-----.,.---11[9------- Each column was divided into the sections indicated above and the corresponding eluates were vombinfJ<l. the petrolewa ether solution f'rom SectiQn I was ehromatographed on two coliumna and developed with 10% acetone in petroleum ether solution. The two main zones were identified in the visual spectroscope as all-trans• lycopene and neolycopene A. the petroleum ether solution or Section II was chromatographed on eight columns and developed with 6'1, acetone in petroleum ether. Af't.r development the following sequence was observed. 4 brown 12 pale red 113 dull orange Section V Section Vl ---·-------~---------20 bi-ight yellow 54 yellow-orange ____...... 4 red-orange 10 orange ,30 colorless --------·---------•-------·--- The sectior..s ind!cated were cut and the eluates oombined. A test in the visual spect,roscope with iodine showed that only Sect.ions V and VX contained poly•W""lycopenes. folr-ea1-l;:rsoptpe I - The petroleum ethel," solution from Section v was recbromatographed on one column and developed with 6J acetone in petroleum ether. !he solution of the main zone was evaporated to dryness in •acuo and the residue was dissolved in a minimum amount ot warm benzene and transferred to a centrifuge tube. The pigment was ctystallized by adding absolute methanol dropwise and cooling until crystallization started. A total of f'our volumes of methanol was ad<led. After cent.rii'uging and washing twice with methanol, the crystals were dried and recrystallized as above, Washing the crystals with hot methanol and decantation ot the mother liquor was necessary to get rid or colorless impurities. The third crystallization gave a material which was analytically pure. AD!l• Oalcd. tor e40H56 : C, 89.48J H, 10.52. Founds C, 89.6.3; H, 10.5?. Yield or pure crystals was 9 mg. A solution cf the crystals in hexane showed the same maxitna and minima as pol7-2&!•l7copene I which was isolated earlier from P,yncantha. A portion or the material was catalyzed with iodine and the main pigment zone was separated on the Tswett column. This zone did not separate from an authentic sample of lycopene in a mixed chromatogram test. Polr-.saa-J.7copene III• The petrolellJ'll ether solution from Section VI .- was chromatographed on one column (alumina•oelite 4: l) and developed with 75% benzene in llgroin. The solution from the main zone was evaporated to small volume, diluted with petroleum ether and developed on one column with 6j acetone in petroleum ether. main zone was evaporated to dryness in vacuo. The solution trom the The residue was dissolved in the minimum amount or warm benzene and transferred to a centrifuge tube. Crystallization was carried out by adding absolute methanol drop• wise with cooling. After three ceystallizatione the yield wae 17 11g. AB!• Cal.ad. for C4off56 • C, 89.48J H, 10.52. Found.a C, 89.Y/J B, 10.64. The absorption spectrum waa identical with polJ'-!1!•1.ycopene III which was isolated earlier from f:aacan~bA• A portion of the crystals was catalyzed with iodine and ohromatographed. The main. pigment zone was separated and a mixed chromatogram test made with authentic all•s:!!!i• lycopene. There was no separation. Proly;goJJ!pe - The petroleum ether solution of Section III was chromatographed on six columns and developed with .3• acetone in petroleum ether. Each column showed the following sequence or zone,. '3, brow,n 125 pale bu.ft 75 orange Seetion VII 30 bright yellow orange 8 orange S yellow 3S colorless A test in the visual spectroscope showed that Section VII was the only zone containing a poly,..!&a•lycopene. The solution f'rom Section VII was evaporated to dryness, dissolved in warm benzene and crystallized bf adding absolute methuol slowly with cooling. After washing twice, ,..,.. crystallizing and washing twice the ;yi eld of homog•neous C!lr,stals was 585 mg. The absorption spectrum identified the piguu,nt as prolycopene. A trial. chromatogram of the solution frea Seetion IV showed that it did not contain any appreciable amount ot poly•s!l~lyeopene isomers and accordingly it waa not wo~k•d 9• D. PREPARATION OF THE MATERIAL FOR BIOASSA!S fhe isoJ,ation of a11.. tra.ns- l •carotene, neo• '-' ...carotene P and pro- ~ -carotene was deael"ibed on Pages 67 to 71 or this Thesis. A few milligrams or the analyzea crystals were dissolved in peroxide-free absolute ether, evaporated in e. stream of 002 and dissol-ved in Wesson oil, £Qr the extinction curves and for the animal tests. Mixed neo~-carotenes. 12 mg. of lS' •carotene in 50 ml. 0£ petroleum ether was r•tluxed in an all-glass apparatus, in diffuse daylight for½ hour while nitrogen bubbled through. The solution was then developed on a 22 x 4.4 cm. calcium hydroxide column (Arrowhead Lime Products, inixed with celite 3, l} with petroleUl)l ether containing 2%. acet.one. Below the orange-red main zone or unchanged all•trang torm, a heterogeneous, much lighter zone appear$<! which was eluted with methanol. This eluate was kept at o0 o. While the upper sone was re•isomarized and chromatograpbed. After 4 such isomerhations, the combined mixed neo• ~ •carotenes were transferred with water ftom methanol into petroleum ether and freed by chromatography f'J,om a small amount of all•trans contaminant. The main zone was then eluted with perox1de-£ree absolute ether and diluted to 150 ml. From this, 120 nu.. was evaporated and dissolved in Wesson oil while three 10 ml. samples were necessary to estimate the concentration, as follows. Each or two such aliquots was evaporated, dissQlved in 40 ml. of petroleum ethei-, refluxed for 30 min. as above, diluted to 100 ml. and estimated in the Beckman spectrophotometer on the basis of earlier data. The third 10 ml. d.i4uot, after evaporation, was dissolved in Wesson oil and extinction curves were taken (Figs. 26 and 27J see also Table n). During the bioassay period the oil solutions were kept in dry ice, in darkness, and showed practically no change in the extinction values. Details ot the reeding experiments can be found in reference (8). Table II Molecular Extinction Coefficients of Some Stereoisomeric •0.arotenes in Wesson Oil at the Wave Length of Maximum Extinction. Wavelength (B91) ill•k&n@• ~ •carotene 473,;.4 1:3.1 Neo- 'is-carotene "i 471•2 11.2 Mixed neo• c5 •carotenes 466-7 9.5 Pro• K•carotene 468-9 10.s All-~- ~ •carotene was prepared from pro- ~ •carotene by the following procedure. 150 mg. of pro• ); •carotene was dissolved in 80 ml. of petroleum ether in a 100 ml. pyrex volumetric tlaek and 4.S mg. ot iodine dissolved 1n ligroin was added. The solution wras mixed and then irradiated with a 400 watt, water cooled projection lamp bulb at a distance ot 10 Clll• tor 5 m1ns. The fiask was shaken several times during the irradiation to mix the contents. The solution (a small amount of precipitate appear-4) was chromatographed on one column (Sierra Lime-celite 3 i l} and developed with 6% acetone in petroleum ether. The main, upper, red zone of all... trans- ~ •cal"otene wad eut and eluted. The lower pale zones of neo'6 •cai-otenes were cut, eluted, and their petroleum ether solution was -88- 12 - - - ~ - 10 \ I I I - - I I - \ ~ - - \ I \ - \ \ - - 2- I / I I I I I 400 I \ \ \ \ \ \ I I - \ I I 4- - \ I - I \ I - I I 4·50 ,.- - - I I I I - 11 11 II I _ I 1 _, - - i \ ,, I 500 mp: figure 26. r~olecular extinction o~es ot all•~rw• r' - ear~~ne (full lihe) and pro- t. • C$roten• (dashed line) ~ Wesson oil. Wavelengths in '1• 10 /~ I \ ts ,c _: f= Oo f=_ I \ I \ ,-.,I w v I \ \ \ \ 6 I I 4 \ /\ \ /i \ \ \ 2 \ \ 400 450 ' 500 Figure 27" Molecular extinction curves in Wesson Oil of neo-- )' - carotene F (i'ul.l line) and of mixed neo• 't •carotenes (dashed line}. Wavelengths in D1)1• -90- treated again b.Y the procedure •just described. The combined eluates of all-trans- ¥ -carotene were trans£erred to petroleum ether, washed and dried. The petroleum ether solution was diluted to a composition ot petroleum ether, benzene: 9 al (total volume ca. 100 ml) and chromatographed on a 22 x; cm. column (alumina•celite 4 s 1). The column was developed with 60%. benzene in petroleum ether. A.fter devel9pment, t.be main zone was cut out, eluted, transferred to petroleum ~ther•'benzene., washed and dried. ihe solution was filtered through a tine sinteJ"ed glass funnel . and evaporated to dryness in vacuo (water bath at 40°). The residue was transferred to a 15 ml. centrifuge tube with the minimum amount of pure bensene and crystallized by adding absolute methanol dropwise., w.1.th stirring, until crystals appeared. More methanol was added untU ratio of benzene to methanol was 1 a 4. The centrifuge tube was cooled in ice-water m1Xture for three hours, centrituged and mother liquor pipetted off. The crystals were washed once with-benaene•methanol l a 4 and then with methanol. Af~r drpng, the crystals were redissolved in benzene and recrystallized twice by the above procedure. The final crystals were dried 2 hours in vacuum at the temperature or refluxing acetone. Molecular extinction values were ealcul.ated from two independent weighings" Founds _mol. . . _J.. .11,lem..; 15.7 x lu-r at 461 mµ in hexane and ~:;: =14.2 x 1o4 at 473 mp in Wesson oil. Ana};. Caled. for C401i56 a c, 89.48; H., 10.52. Found, C, 89.79; H, 10.38. m.p. (eor.): 1.36-13'7° Berl blc,ck. All trang• ~ -carotene ~ tomato paste • This material had been saved from an earlier experiment. The chromatographic purification and crystallization followed the procedure just given. ·Em ol.: 15.9 x 1o4 1cm. at 461 111)1 in hexane. m.p. 129-130° (cor.) Berl block. In a mixed chromatogram test th$re was no separation of all•trans• cax-otene .I! pro- l .:.carotene and the i •carotenes from tomato paste or eommercial carotene. The values for the Molecular Extinction coefficients of all-trans• ~ -earoteru, in both Wesson oil and hexane are higher than previo\1$11 reported (this thesis, ,9) which would partially account for the higher activities found in the rat tests. fto- ~ • carote41 - The isolation was deseribed on Page 67 or this thes1e. The extinction co.e f.ticient in Wesson oil was I E~~;: =10. 8 x 1o4 at 468 DJl• This is identical with that reported earlier (Page 87 of this thesis). Il.i COLORLESS POLl'.BNES A. lSOLATION OF PR!tOFLUENE FROM JACARANDA 920 g. of fresh blossoms•£ ataoaranfla 9vali!nia R. Br. were collected in June, 1948. The, green stems were removed and the blossGl!ls were covered with methanol. I-t was net necessary to gr:bid the material.. After standing OV$"1ight, the methanol (:oa 3._S liters} was draUled ett, mixed with a liter tt petroleu. etht~· (b. p. 60- 7rl'), and water added cautiously to transfer .iraeted -with 500 ml. or petroleW!l ether and then disc,a rded, petroleum ether solutions were combined; washed, and dried .. The The filtered solution was conc•ntrated in vaeuo to ,a volume of 100 ml• This solution was cmtomatographed on a 25 x 4.5 cm column {Ca(OB) 2 - Shell Brand) and developed with ligroin, The tollowing sequence was observed on the column b"om top to bottoa (Figtu-es on left denote width of zone in mm. )c :'3 fluorescent 1; empty 7 tluoreseent 15 empty 8 yellow and nuoreseent 20 empty a tluorescent 3S empt1 8 orange Section II 70 nuorescent Section III Curves of all the tluore,seent zones wre taken in the Beckman spectro• photometer but only the solutions from Sections I and III showed a characteristic spect"'1. The CUM'es gave almost identical positions or the maxima and minima (corresponding to phytof'l.uene) and the,- were estimated together using published values of ~cm. tor phytofiuane (72). The total ameunt estimated was o.;2 mg. tor 920 g. or o.6 mg. Pel" kg. ot fresh blossolllB• Section II gue maxima at 478,450 mp. and was estimated as ~-carotene using the ~ 01 • value of 13.9 x 1o4. Total p •carotene F -icm. . was o.o, mg. or o.06 mg. per kg. ot fresh blossoms. -94S. ISOLATION OF PHITOFLUEiOL FROM TOMATO PASTE 30 kg. of commet¢1al canned tomato paste (Caapania - lest Coast Packing Corp., Long Be.a ch, Gal.if.) was the starting material. Each 3 kg • po~tion was shaken with 4 1 .. of 9~ methanol tor 20 runs. and filtered through a cloth in a basket centrifuge. The aqueo'U8 alcohol tiltratf from each .fraction -..s extracted with J. l. of petroleum ether and th• aqueous phase was then discarded. w~ked up in 6 kg.-portions. The partially dehydrated paste was Each portiou was shaken for 20 111ins. with 4 1. or methanol and filtered through a oloth in the basket centr1fug•. the filtrate from e~ch 6 kg.-portion was extracted with l l. ot petrolea ether by adding water cautiousl;y and swirling .. If necessary the aqueous lay•r was extracted once more with 11. of patroleum ether (this was done if a small test indicated that fluorescent ma.tttial wouid pass into the ligroin phase). The aqueous phase was •then discarded. The partially dehydrated paste was worked up in 6 kg.~portions by shaking each portion with 3 l. of methanol ♦ 3 l• of petroleum and filtering as above. This extraction was repeated l times tor eaeh portion• &f'ter which the fluore• scence of the l:lgroin extraet was very slight• The combine.d pet.roleua ether-methanol fraction wafl poured .into 20 1, separatory funnels• water was added cautiously and the nuor♦scentmateti.al was transferred to the petrole'Ul'll ether phase>, The total ligro1n solution was washed free of methanol; tlr.ied and filtered, 'l'he filtered ·sol.ution was cone$ntrated in vacuo (450) to a total volume of 3 l. '?his concentrate was sapenined ov.-.rrdght with 2~ methanolic potassium hydroxide. Alter washing the petroleum eth•r solution free or methanol and alkali the precipitate ot iUDIDl1' crystals was filtered off. This precipitate was redissolved in benzene and recrystallized giving crude lycopene. The mother liqUQrs of crystallization were evaporated to dryness, redissolved in petroleum ether and added to the bulk of the saponified solution. This total solution was filtered through a 7 cm. layer of celite in a sintered glass tunnel (12 om. diameter) to get rid of gummy material which would inter.. fere with chromatography. The celite -was washed with petroleum ether ·· until the carotenoids and fluorescent materials had passed through. · • ,i •· • . . .• : ($ome fluorescent material is adsorbed/ 'on the celite, but is discat-ded). The ligroin solution (5.41 .. ) was ehromatographed on ten columns (Sierra lime• Oelite 3 al) and developed with 10% acetone :1n petroleum ether. Ea.eh column showed the following sequence (Figures on left denote width or zones in m.)1 22 orange•brown 15 orange. 4S red• l;ycoptn• 10 yellow-orange 7 red-oran~e t contains t •carot-me 12 yellow 35 orange; ~O t Section I• fluorescent ~arotene . m:tit~ oran.ge and yellow zones; fluorescent J>h)'tofiuenol was not well separated at this stage and Section l included the lowet halt or the neolyc.open• A zone and the upper half ot the 12 mm. yellow zc,ne• The combined petroleum ether solutions troa sections I were recbrome.togt"aphed on four columns and developed with 15% acetone in petroleum ether. The £o1lowing sequence .was observed from top to bottom, 7 colorless 13 red 25 red, lycopen.e 48 orange• .neelycopene A 6 yellow 17 pale orange Section II, fluorescent 2; red-orange 40 yellow 10 orange 30 •mixed zones The petroleum ether solution from the combined Sections II {containing the phytotluenol) was elu!omatographed on eight columns and developed with 1S% acetone in petroleum ether giving the following sequences 20 colorless 40 red, lycopen• so orange, neol.ycopene A 30 pale orange 2; orange 30 y-ellow, phyto.f'luenol 25 orange 60 colorless -97- The phyto.tluenol zones were combined, and their petrole'U,11 ether solution was chromatographed on one column (alumina•Celite, 4 s 1) and developed with 3~ acetone in petroleum ether. The main, fluorescent zone was eluted, transferred to petroleum ether, washed and dried. An es-t imate ot the amount (12 mg.) was made using values of tor ph;y-tofluene (54). Ii~. published Evap.oration to dryness gave a slightly- yello.• oil and attempts to crystallize it failed. Best values obtained with •~.d.ghtxi samples gave Ei!m.: 410. If the moleeular weight ot phytonuenol 111 great•~ than pbytoflu.ene by- one oxygen atom {i.e. c4rJI64o) this would give .a purity- ot 35% tor the sample. An attempt was made to prepare an acetaw: by dissolving l mg. of phytotluenol in a few dl"-ops of pyridine and adding l clt'op et acetyl chloride. Subsequent chromatography and tests or the tlu.oresee!lt material indicated no change in partition coetticient ·a.n4 adsorption behavior before and after treatment with methanolic potassium hydroxide• Partition coefficients between hexane"' 83%(v/v) ethanol; and hexane"' 95j methanol were carried out in the following mannel". A solution of phytoflaenol j,n hexane was mixed with a roughly equivalent vUume or the aqueo\1$ methanol (or ethanol) and shaken in a s.e paratol'y tunnel. Alter the phases separated, equal T<>lwnes ot the upper (,J,i) and. th~ l,_ ower (~) phases wel'e taken. Hexane was added to LJ then both tJ and L nre wa.s hed f'ree of alcohol and dried. th• filtered solutions ot U and I, (in hexane) ••:v•--d1lutedto equal 'Volwae and the extinction values determined in the Beckman spectropbotometttr at the main maximum. Partition coet'ticient : C (upper) / C (lowe:r} E (upper) / E (lower). = Values tor phytotluenol obtained ar• 2.2 Oiexane- 9S% methanol) and 6.0 {hexane• 8)% ethanol). III. SYNTHESIS OF STERE0IS0MERIC DIPHENYLBU'.l.'ADIENES Lisbt Sourceg .IW! Conditions g! Illumination • Chromatograms were inspected in the light of a General E+ectric Purple X bulb using a moulded Cmrning light filter No. 5840, 0.25 inch thick; "Mineralight ~l" was less satisfactory due to its different light filter. Iodine catalyeis (in hexane solution) was carried out using amounts of iodine equal to 5% of the weight of dissolved material and illuminating for 10 to 20 mins. Under the swne conditions benzene solutions should be illuminated for an hour. For artificial illumination of iodine•free solutions a 250-watt Mazda olear projection lamp bulb (Code -250 T 14/3 ..12ov) encased in a water jacket used {distance from filament to center or Pyrex volumetric flask, 10 cm.). Transparent quartz test-tubes were found satisfactory for insolation experiments (end temperature., 20-25°). %hermal treatment• Refluxing of hexane solutions (5-20 mg. per liter) was carried out in darkness., in a slow steam of carbon dioxide. In the melt experiments, 4•12 mg. weighed samples, in evacuated and sealed pyrex capillary tubes, were submerged in a dibutyl phthalate bath (205-210°), in darkness. After ten minutes the tubes were rapidly cooled in water and their contents examined without delay. A. trans•Diphenylbutenine C6H 5CU:CHCIC06H5, was prepared (in a crude state) from phenylacetylene-cop~r according to Straue (47). Purifiea tions a solution of 8.5 g. of brownish, crude crystals in 100 ml. of petroleum ether was developed with the same solvent on a silicic acid column, 22 x 4.8 cm .. {The f"igures on the left designate thickness or the zones in Jlllll.; n. means tluorescenoe or fluorescent in ultra.violet light): 30 several b~own zones (in daylight) 30 white fl. (1 g. oil) 7 brown (in daylight) 8 interzonet no r1. 50 white fl. {lg. oil) 80 . column-fl. quencheda Filtrates main product non. The main zone was cut out, eluted with ethanol, transferred into petroleum ether• dried with sodium sulfate and completely evaporated in vacuo. A solution or the slightly colored crystals {5.5 g.) in 50 ml. or hot methanol deposited 3 .g. or white needles in the eold room; m.p. 96°, cor • .An additional lg. can be obtained by chromatographing the mother liquor as above. -100- B. cis•tranp-Diphenylbutadiene from MfAAS•Diphenylbutenine. In an all-glass apparatus (with mercury•sealed stirrer} the solution of .3 g. or the butenine in 250 ml. of alcohol was 'rei'luxed in darkness with 2 g. of zinc•oopper dust (from copper sulfate and zinc; washed with ethanol) and another 2 g.-portion of the metals was introduced -after thirtr hours. The sharp extinction maximum (originally at .306 mµ; in petroleum ether} gradually disappeared and, after one hundred and ten hours of ~efluxing, the smooth maximum of sa•trans•diphenylbutadiene was observed in a. small sample. Then 250 ml. of petroleum ether and 1 liter of water were added to the filtered liquid; the aqueous layer was r&!'extracted and the total petroleum ether solution was repeatedly washed and dried. The following chromatogram, obtained with the same solvent, on a 28 x 8 cm. alumina column, refers to one third of the crude solutions 30 several, partly n. zones 25 blue fl.: trans-tran@•diphenylbutadiene 140 fl. ot alumina quenchedi crude .2i§•trang When the bottom of the lowest zone was 7 om. from the end of the column, the quenched section was cut out in two halves of which the upper one contained unchanged butenine (its extinction curve was not lni'luenced by iodine). The bottom half of the quenched zone showed the spectroscopic character of ~-trans-diphenylbutadiene, and a sample yielded (after iodine catalysis in light) the f-'ne structure of the trall§_... trans form. This fraction was rechrornatographed as above. In order to eliminate the last impurities, especially for optical --101• observations, 3 mg. of the eia-trans compound (in 3 ml. of petroleum ether) was adsorbed on a 20 x 1.9 cm. column (alumina} and washed in the same solvent. Each 10-ml. portion of the (non-nuoreseing) filtrate was tested as follows: A few drops were catalyzed with one drop of iodine solution (0.3 mg./ml.) and the test-tube exposed to the 250-watt lamp .from a 10 cm. distance for one to two minutes. If the sample showed fluorescence in ultraviolet light, the traction was kept, otherwi1e rejected. This test was positive between the fractions l 7•29. Fractions 18-28 were found spectroscopically to contain pure £!I-trans compound; 3 g. of the butenine yielded 0.75 g. of the purest product. The molecular extinction coefficients were given earlier (73). &!!l• Calcd. for c16Hl4: C, 9.3.16; H, 6.84. Founds C, 92.72, 92.89; H, 6.84, 6.88. C. Ci§•Cis•Diphenyl butadi ene cis•cis•Diphenylbutadiene was prepared by catalytic reduction of di~henyldi aoetylene, C6H 5c:cc!cc6H5, by Kelber and Schwarz (26) as well as by Ott and Sehroter (41) in yields of 8-32%, depending on the catalyst. Although our yield remained between these limits (tor examplet, 24%), we recommend the following isolation which is based on spectro• soopic- control or the reduction process and, chromatographic purification of . the product~ During the reaction the triple bonds are reduced not only to double, but in part also to single bonds. These competing processes can be rollowed by the changing extinction curve. The sharp and high peaks or diphenyldiacetylene at .306 and 326 "1• (in hexane) gradually disappear and the much lower, smooth maximum of cis•eia• diphenylbutadiene at 300 mp. then makes the main contribution to the curve. A satisfactory amount of the latter compound is clearly indicated if a small sample (diluted with hexane) is examined in the spectrophotometer before and after iodine catalysi$ (in l ight). The new maximum (now at .328 mp;.) whould be at least 1.5 times higher than Emaz. was before this catalytic treatment. The following operations should be carried out in dim light. A solution of lg. or diphenyldiacet;rlene in 100 ml. of 95% ethanol was shaken with 0.25 g. of palladium•barium sulfate (40) until roughly .350 ml. of hydrogen was taken up within one quarter to one half hour. Arter filtration, "'the--material was transferred with wa~er into petroleum ether and the aqueous phae~-re-extracted. The eombin,ed petroleum ether solution, after washing, drying and concentrating to 50 ml., was developed on the alumina column (30 x 7.5 om.) with about 2 liters of the same solvents 6 dark {in uviol. light) 8 greenish fl. 20 blue fl.: trans ... trans.,.dipheny'lbutad:1.ene 16 non•tl. interzone 60 eolumn-tl. quenched: cis-:g;ans•diphenylbutadiene 26 interzone (borders blurred) 50 column-fl. quenched: c1s-s,u.•diphenylbutadiene llO empty section The location of the 4!.!•ci1 compound possibly oould be improved by using- a strongly fluorescent column as proposed by Sease (45) and Brockmann(J). The m•.w zone was out out, eluted with ethanol, transf'ened into petroleum ether, washed, dried and completely evaporated tn vacuo. The oily residue was then transferred into a small centrifuge tube and dissolved in a minim.um amount of warm 95% ethanol. Crystallization was observed at room temperature, whereupon the tube was kept at 4° over• night. Yield, after recrystallization, was 243 mg. of 2is'• J!ll.•diphenyl- butadien♦ which showed distinctly difte~ent the :!trf:D!•trann isomer. crystal to~ms tr.tom those ot The extinction coetficienta are given in Table IIJ;. AD!l• Oalcd. tor <li.6ll14t C, 93;,16J ll, 6.84~- Found., C, 93.13; H, 7.17. •104- Table XII Molecular Extinction Coetficients or s!l•oi,Diphen7lbutadiene at the Maxima (ita.licize~. and .ldnima cis-m form Emol. lem. •. • Solvent ••· Hexane Benzene !71 Mixture of Stereoisomers upon iodine catalysis ol. ~ cm. X 10•4 X 10•4 229-300 2.96 344•%2 3.40 251 0.51 340 3.35 328 5•.36 318-319 • 4.50 Jl5•316 4.56 255 0.20 230-231 1.36 l52•J5l 3,17 Jlt1. 2.9; 334•3,3~ 4,.85 325-326 4.00 3?).•322 4.05 - 306 2.67 Spaj.J.•§O@lS! Separation ~ the Three Stereoisomer3tc piphenzl- bytadienes - A solution which contained l mg. of each isomer in 10 ml. of petroleum ether was developed with the ~ame solvent on alumina (20 x 2 om.); the developer was forced through Lthe column by- nitrogen pressure in order to a.void partial evaporation of the filtrate. T:h• fluoreseent :trw-t:!'M@ compound remained near the top. Small samples ot eaeh traction collected from the filtrate were submitted to the iodinecatalysis and tluor-esoenoe test as described. The first 120 ml. or the tl~ .-e found to be tree of substance; subsequent 5-ml. fractions were tested with the following result ( 11 eis•cis" or nsi!-tg:anptt refer to the eontiguration before :iodine catalysis): No. 1•7 strong fl., cis•eis 8•9 weak fl. i m•c,:tg 10-12 almo$t no fl.1 traces 13-21 very strong fl., gis•trys 22-26 strong n.a cie-trano 27•30 weak tl.1 cis ... trans 31 non. The tractions were also tested spectroseopically. -106References 1. 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ZeohJnsister, L., and Pinckard, J. H.: Experientia It, 474 (1948). 64. fschmeister, L., Pinckard, J. H., Greenberg, a. M., Straub, E., Fukui, T., and Deuel, ll. J. Jr.: in press. 65. Zsohmeister, L., and Polgar, A. I J. Am. Chem. Soc. M, 1856 (1942). 66. Zechmeister, L., and Polga:r, A.I J. Am. Chem. Soc.~, 1S22 (1943). 67. Zeehmeister, L., and. Polgar, A. I Science 100, 317 (1944). 68. 1.achmeister, L., and Polgar, A.: J. Am. Chem. Soc. ~ 137 (1944). 69. Zeohmeister, L., e.nd Polgar, A.s J. Am. Chem. Soc. 92, 108 (1945),. 70. Zeobmeister, L., and Sandoval, A.: Arch Biochem. l!, 425 (1945). 71. Zecbmeister, L., and Sandoval, A.: Science 1:Q!, 585 (1945). 72. Z&chmelster, L., and Sandoval, A.: 73. Zecbmister, L., and Sandoval, A. z J. Am. Chem. Soc. 9,2, 553 (1947). 74. zechmeister, L., and Schroeder, w. A.; Arch. Biochem. 1, 231 (1942). 75. fachmeister, L., and Schroeder, w. A.a J. Am. Chem. Soc. §!!, 197 (1946). J. Am. Chem. Soc. 21., 117.3 (1942). 76. fschmeister, L., and Schroeder, w. A. a J. Biol. Chem. lUt., .315 (1942). 77. Zechmeister, L., and Tuzson, P. 1 Nature 1'!, 249 (1938). 78. Zeclme:tster, i ., and ii:ms.on, P. • tioohem. J . J&,. 1305 (19.3$). 79. Z'Jcl.'lzn$1steJ-, t ., and Tuzson, l>. t Ber. Z&, :t.340 (lCJ)9). so. f+eehneiste~. L~. Wille, B., and Pinckard, J. it. 1 3. Am<i Che1n. Sec. 72, 1938 (1948). • lll Propoeitions submitted by i . H. Pinekal'd 1. $ ~ ':Lt has 00,f;'fn ]')l'oposed that ste:rie hhidrance of ceit-tail'l double bonds .in oa:t'otenoids will prevent a eia <:onfiguration at th!$ bond, and sinqe this is based on th& concept of coplanarity, it would i» of value to know th$. %'8$ona.nee energies of oarmn.oid.s, and the 4ii'feJ"8IJCt in sMrg1es ~tween $'tereois0Il1Ell'ie .forms or earotenoide. The determination ot these ~~rgies would be possible tbroiugh (a ) Heats of }¢rogenat:ton, and (b) Ue.ats or combustion. L. la..Uling, Fortschr. Ohe.m. Organ. Naturstoffe Jt 203 (19.39). o. B. Kistiakowsq et al. J . A. o. s. g Uh; (1936) R. : . Corruccini and E. c . Gill:~n•t, 3. A. o. s. §l. 2925 (1939) 2. 1h the o~rsion of provitW'flins A to viitamin A1 1n bio1Qgic£\l systems, theN." is a e,paeifio attack on the ~ntral double bond of the molec~. Since the atrangement of mtbyl grcups around ·Uds double bond is dttfe%'.ent from that of other .double bonds, I propose that proparly substituted diacyl peraddes of the type fl,_ fl 9, {JR, c-o-o-c.-~ R. 1, R,, ~ 1 w<mld attack t his central double bond in preference to <>ther$. 3. Since it ts diffieult to obtain po.ly•a!a forms or polyenes, eithG:r by direct synthesis; 01' by isolation from natural sources, the two following nethods -o f produeillg sueb f'Ql"ll?S shou1d oo investigated: -112... a. A.ppl.ioation or very high pressures with mod&:r.ate heating. b. Xrraet.ation with monoe1¢omatic .llght, or light which i$ suitably filtered to $llmina.te carte.in frequencies which would promote re•ison.erization. 4. I propose that a study of the effect of ethylene on the ri~ning processes i..'1 aome. fruits should be carried out with c14 laoolled ethyl.en$, followed by isolation of some eompounds from the fruit, in order to follow the oour.se of reactions taking pl.ace. during ripening. ;. The affect of compounds w1 th halogen•lil<e proP9rties, such as thiocyanogen, in producing oif-tr§M isomeriza-tion, shQU.ld le studied. 6. Since oarotenoid acQllimllation occurs in such animal tissues e.s the oorpu$ luteum and fat bodie$, !t would be of intere~t, e . g. in the frog, to test the viability of $perm or eggs in carotenoid depleted animals. L. Zechlneister, from :SrgePltlsse del' Pbysielogie, biologi$ehe:n Chemie und. exparmntel.le11 i'ha~ologie, (19.37). 7. The use of more or less boln()gene·ouc0 protein •te:rials, such as oaat)in, :albumin, etc., as adsorbents £·o r chromatographic separation of carotenoids and other substances, should be tested. 8. Some aeetylem.c compounder yield c&@ isooers. of tbe pol,ene preduced by reduoticm. It would ~ of intere$t to eynthssize di$rYl•polyacetylems ot the type AR•(CIC)n•Att, where n is grea:ter than two, and detenn.i:t'w whether stable qi& iscu,re ot ,AR.-(Clt:CH)h-.AR are produced by reduction. This would te. of interest, iince no stable eig isoroors of' higher ~ M s of this t7J.e are known. R. Kuhn and A. iintt,rstein, Helv. Chim. Acta J:!, lJ.4 (1928) .u,;. 9. • $in~ ~ · he&~ter fish accumulate v i ~ ½ in ti. liV&r, it would l:s· ()f va.l • to e&l'l'Y out feeding t1xper1Den.ta, using diff•rent oarotenoi-ds, t .o $89 wh(3ther the1'8 is a NJlation batsmen the kind ot carot.enoid. · .and 'V1t$Jlli.n pX-odumad. lh 4. lior.t<m, Nature m, 69, 405 (1944). :to. :tt h diffieul.t tor studen.t:s to get $1 i&la .of' ·tile rese:ar(:h ·p r ~ ce,n,tttd out 11y variOtJ$ groups in the ebltrnistr.y departr.ent. I );:ll"Gpose • that botmd.vol~$ ~ prepa~d, containing the original papers pu.bcllshed ·t,g ineuibe.r s of this division, and that these vollitnas be placed in the &tpartment library.