cTg M o iC k o 3 ,o i* a

ST i)Bi3 m aariniAiLï ocm km ia pl« « k c îîo is i ,

A Theaie Submitted by Hoi^aa Jum ZWleod

a candidate for the ïiegree of Doctor of

Hiilosopiiy

Bovwbar, 1902 Rqysl Holloway College, ItoivoFslty oT Loadtesi, KneLsfleXd Grom, Surrey. ProQuest Number: 10096673

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ABoTRàOr

?arioud zaonocotyledenolls aad dicotyledeaoue plants have bem examined aM foimd to pooaeaa an msym& capable o f deaminatiJig 3j^k~dlhydro]Qnph%^ (DDFA) id th the formation of trana^caffeie aoid# The activity of tels m s^m In barley» dandeli

Broad^bean leaves also contain a tyrosine deaminase.

It has been shown that ^foreign" phenols Gm move rapidly up and down the stm s of broad-bean plants and that glucoeylaticai of many of taeae cmpçmâa readily occurs* Phmols fed into the out ends of the main veins of leaves quickly appear In the sieve tubes of the stem* This has be^ proved Ifiy the use of the aphid, plsi* A#iid techniques have also strongly suggested that the sieve tubes of broad-bean and «dllow nomally contain phenolic compounds# - 3 -

The réaction of boron trichloride and methanol on cirnianic acid derivatives haa been Investigated in detail and a procedure hoa been devised idiiCi aids the identification of these acids in plazxt tissues* Using tWn method all glycosidin, esterifled and methoxylated derivatives are converted to tha corresponding mono*», di^, or tri*»bydroxy*»met^X cinnamates whidi can be identified on paper chromatograms or electroi^oretograms* Thus the cinnamic acids pmsent can be classified into one or more of those families*

The p€q>er electrophorotio examination of hi#% molecular w ei^t phenols as aso dyo derivatives has also betm Investigated* These phmols have been coupled with diasotioed ^amitiobenzoio or diav»otised sul#ianilic adds* The resulting possess strongly dissociated acidic groupings md tWrefore exhibit relatively h i^ electrophoretic mobilities in cw^parlson with the imsubstituted phenols. Xt lu a# pWmw* to ftofmtat K*

I w>itM #Xao W m %9 Wmk Dr, f • Smia md £• e, of tM Xaw Smwai# far aalpfal

4 l# a u # M o w , «ad $ w X*Q#X* M a#$ mmWeWom, # 1 1 , f « r MwyMlmg aa^liM of fiiagfoaliMu» aial*

X m Indb^toâ to tb# A@^auiwml Eosoorob Coaaoil for the pmvWa* of a gsrau* imitât «*ta W a ww* poool&la. > -

QQtgEîrtS Page to. Abstract •• •• •* «« •• Z Aoknowledgiaant* ,« #» •• >• U Contents •• •• •• •• 5 mtroducbion and Dlsciiselan «• •• 6 (a) DOPA deaalnase .» ,« 7 (b) Transloeatlon of pbeaolios •« 3U (c) Identification of nstorally- oeeurrlng olnnamle aolds wltii BCiynettianol ,# 51 (d) Paper Elaotrophorsois of hig^ mol* wt* ^ en o lle s «• •* 66

Oensrsl tothods •• »• 75 Experimental ** •• ,* 8o References •• •» •• *• US

\\

\ \ ■s ' V'.. \ m ^ m

imaooimim mo discussion CHO 0 - 7 ' CO^H H^-OH I 4- I XIV OH C - O ® H Ç -O H OH H-C-OH HO C H i i w o o @ H —O H LHs, o © \\\ XI OH CO,H XIII HO

vn

rt > C O ,H ^ 0>pL/ ^ OH XXVll HO ^ HO h o / OH ^ _ XXI VIII ZT

y H i c h j - c h CH=CH.COjH CO^H t O i H

4 / 4 /

XX OH OH H OH 4 Xxm

P y H i. XIX CH, .C C H .-n p H CH=»CH.a\H ^■''^COiH J/ i OH OH ^ 0 " XXlV 0 “ - _0 G H i-C: H CH*CH.COj.H xyx do,H I OH XXVl 0 .CH3 ___ H3C,0-^0.CH3^i? XXV LIGNIN CH=CH,COiH C H = C H .C O a H O h _ « . HO OH

OH

O

OH OH

R- CO OH

OH R.C0CH,.CH0H.CH2C0.CH,CqH

R'COCH^'COCHg'COCH^-COaH ii

R CO.H + CHaCC^H + CHg COgH + CH, CO^H i CH. OH j c V,

O 'C H C O ,H

t-IG 1 % p H c C H , CH^

CHOH CO^H - 9 -

ÜQPA DEAMBÎAS£ IH PIANOS

Biosynthesis of Phenolies

There exiats# a t present, two aoœpted pathways lead to the build-up of naturally occurring phenols in plants (see Fig# I)* 1 2,3 V Tbe first is the «aceoate unit" pathway, whereby the production of many aromatic ocmpounds with the resorclnol and phloroglucinol hydro:Qrl pattem a, the flavonoida, is postulated. The compounds arise by head to ta il linkages of acetate units# Using isotopieally labelled acetate it has been demonstrated that the biosyntiieslB of fatty adds in plant tissues / from acetic acid, or acetic acid together with higher fatty acids, proceeds without loss of carbon# For exaji^le, the synthesis of butyric acid (Î ) in Clostridium kluyveri proceeds as follows*

QHyCO^n * CH^gK ------> GM^*CO#Ciïg#COgH

+ oi^.cOgH n g . ( ÏÏ )

caj.caîg.cHg.cOgH ( I )

Ho enzyme studies have been made of the mechanism of acetate assimilation by plants or bacteria, leading to the formation 7 of the lower fatty acids, nor to the role played by comaayme A (3*, 5*-diphosphoad®aosiae-U*'phosphc^antetheine ) in their synthesis# - 10 - iîowever, the synthesis of higher fatty acids (e»g# citric, succJnie and malic acids) from estate units proceeds way of the inter- 20 . mediates of the tricarboxylic acid qyola# Coenzyme A is necessary not only for the aotivatim of acetate, but also for the conversion of all the carbcoQTlic adds into an active form# Ho plausible explanation has be^ found for the chemical mochanism of acetate X aotivatiem because the coensyme A molecule has a nmaber of functianal groups, which make several different reaction mechanisms possible# lyn^ has, however, established that the thiol groD^ is the active centre of the coen&yme A molecule.

The ^keto acid intermediate {IJ ) in the build-up of acetate units is, at present, purely hypothetical, and may exist only as an enzyme bound complex, probably of the co^zyme A e ste r.

g Birch noticed that ini^my naturally occurring phaaols, the hydroxyl groups in tha armatic ring were on alternate carbmi atoms ^

e#g# ring A of flavcmoichi such as quarcetia ( i j j ) *

(È) - 11 - The first biochemical evidence to sxqpport the theory that naturally occurring aromatic compounds arise hy head-to-tail condensations of acetic acid molecules was provided by Birch, 9 Massy-Westropp and Moye Wien they found that Pénicillium griseofulvin formed 6-methyl salicylic acid (jv) from C-carboxyl- labelled acetate (Fig.nj ).

CH 3 CO&H ^•CH^.CO gH (i5) * » OH

The pathway for the formation of a phloroglucinol ring is shown in Pig. I , where R.COgH can be any naturally occurring acid such as a fatty acid, a terpenoid acid, a branched chain acid or a cinnamic acid. The formation of resorclnol derivatives can be eaqilained by reduction of a carbonyl group, not involved in cyclisation, in a non-aromatic intermediate, ihis is more likely than the reductive removal of a hydroxyl group from the benzene rin g . Introduction of more oxygen atoms ortho- or para- to existing oxygenated substituents could then take place without difficulty.

In the biosynthesis of higher molecular weight compounds such as polyisoprenoid conçHDunds, mevalonic acid (v ) has been found 10 to be a direct precursor and, more recently, attention has been focused towards establishing the intermediates between acetate and - 12 -

11 mavalonato. Caie sudh Intermediate 1$ thoii^t to be > iiydroxy-S-methyl-glutaraldehy^ acid ( ^ .

A s ecoaad pathway for the formation of phenols arose 12 from the observations of Davies who, in 19$k, published the results of workon intermediary metabolism in mlcroorganisois* 13 / He established, by the method of imitants that shifcindic acid (VH) is a key intermedi&te in the biosynth^is of such essential arcsaatic smtabolies as #anylalaoine @> ), trsptophan (IV) and _ 14,15" £-axûinooensoic acid

^ C H

ShîMmic acid (^0 is .first forsied from the condensation of a tetrose (D-@rythrose-k-phosg^ate ]) and a trlose (phospho- enol pyruvate [^l]) by way of a 2-keto-3-deoxy-D-arstoheptshikimic acid (# ). A Oj side chain, arising ùnm pyruvic acid is then added to an activated phosphate derivative of - 13 - shikirüiic add @4 with the formation of prephonic acid (M} via tha intermediary The chemical etructure of Z\ has not been I S t3 l-Vt-ÉOU-C positively idmitifled although it is thought to bo SKipro «ranged form of prephaaic acid. Prephenio acid is the branching point of the system# It is unstable and readily decomposes (non-enzymioally) to phenylpj.'Tuvlc acid ( ^ 1 ) or to j^hydarocsy^enyl-pymvlc acid « There la no reason to bellm^e that there is a pathway from phenyl- pyruvic acid ( yVI» ) to tha corresponding jg-hydrcogr derivative (%Tx)# Cy C/Lo LuLxcuiAjL I « C OoC^oL The presence of these varteus in^ ermediates sho^m in Fig# 1 was i/L tvi^kjCA fiXoiA^k> *19 confirmcd^by Hasagawa et al# ' also showed that ^ilklmie aeid aBd~cyçl,ohftyang. r^rhoTryllo acid deM^yatives had-^idosproad aiatrib ttti^ la plant tiasuos#

37 HcOalla and Meish caiTled out e^perim nts feeding shUdLnoio acid to S alvia spleadaus# i>ubsequent s%aminatj,on o f the plants showed tjmt both plienyialaniae ( W ) oad tyrosine had been 22, 23 synthesised# This, and other reported studies showed that l*«nylpynivic acid ( ) and g-t^droaypa@nyl#Tuvlc acid ( XIX ) can both undergo transamination reactions with the formation of phenyls alanine and tyrosine, respectively# Tÿrcsinc ( @ ) has an alternative pathway via mzyrxLe hydroxylation of phenylalanine (vm) and 3,k-dihydrwy "imenylal^ (üo?A-) can be formed by tbe_ 2S XXI hydroxylation of tyrosine# A pathway to cinrueiio acid^\from phenylalanine ( ) via piimyllactic acid ( ^ 0 - Fig. ]V was put forward by Welsh. 11 CO,H

CH.- CH c h ^ - c m CH=CH.COiH \ OH

(wT) ( H ') ( #__ ) la 1961 , Kfflakol aad Com publlahad results showing that iteiylalanlne could a3#o be c^mverted to olimmio acid (^1) by direct dcaninatitm using enzymes from Grsmineae 2*3 md Leguminosae# Heish later dls^wmred an mayme, tyrase, which effected the oonversi

An alternative pathway for the formatiez of the phenolic cinnamie acid derivatives involving hydroaylatim and methosylaticfi, was put forward earlier by McCalla and Heiah in 2.9,30 2$59m Th^ found that ^comaarlc add c

A (aüwja la fig# i ) to tho 21 ocwun'^iug ooid# ^bs w , ostod Oj^w# im 1958 Î1» i^corted tàe o* protoogto^^iG «cdd (xiyTi) ilrw ^ktndc a$M ( W) @n# ^fo y#mat]y 31 ____ is â ite hw aug^^ôGWd tW t «sid ( XltVIH) bo foammd the dlm $t exi^mtlGR of saSMteie &CI4 vltW at loam of tte ù im tio m (?%# Î Î#

^th tW aoetmt# aW idiildmie aoid mm rotpisrod for the fermotlm of phmolloo W,# a wmle;m# IW@o om#oimd» mmm e f miWar to toe çàrni^ 33,34,35-. ai kto#m. Bomml gm## of w^pwera Wre emndedi oiaè / «#erWKA* aalBg iaeta^eaU r %aWUW wwpoaaii ralafcad io sCüMnrt&e «sid, pW^riatmlM aaâ aceâi* wM* Sa tîd* «gy* it Wi taea absMi tte i W% ^ fl&vwol, ^lareetâtt {II] > m i th# ^ ^KWoymWm, qyanidln (vünQ^^ ara fomad ty a @a#laa&lm of tim «MB bioabaaleal {Mttafaya. ^S-Waallad WdLklwüe ssid ( g- maaarle aaW {^ÔÏÏ)» pfe«grlaia»lBa CSM) *ai eisaaaie ludd (g |) «m aU, «at «a preos^ors o£ ta» portiaa of ^larootia la teaetefaeat, OH

HO XXIX - 16 -

there le no evldeaœ^ at present g as to Wiether the hydroxylation occore before, or after Ufâ foimticai of the nucleoe* Ring A arises fr m acetle acid as was show by feeding ^CLmethyl- and carbo3grl*lg^X.led acetic acids#

the different classes of ocmponnds once formed in the plant are o^>able of interoonversion by oxldatieai or reduction of the moieties# the details of these inter- conversions are s till stjeculative as intertransformation bebwen the main groups of flavonoids in the plants has not so far been shown#

Once the pathways to the sia^ler naturally occurring phenolic and otoer aromatic coapounds had been satisfactorily examined, a multitude of questions oomeming their transformation 39,3p, VO 41 to more cce^lex products, such as lignin, %»re raised# Hibbert suggested that siu^ile precursors of lignin were Interoonverted by reactions similar to those in the respiraticm of carbtoydratea# 27 Neito considered it move probable that the pNmolic cinnamic acids were intermediates, since they were so widely distributed V2 in tra c e amounts in plants#

Experiments with ^Omlabellsd c«8i^x)uad3 substan­ tiated tha idea that #em>lic cinnamie adds were intermediates in lignin formation* Conversion of lUfirlabellftd tyrwfrfe to - 17 - lignin occurred readily in certain plant species, such aa 29 members of the Grmlneae. üeish suggested that a tyrase (tyrosine deaminase) was necessary for one stage in the conversion of tyrosine to ligg&in#

Aoerbo ^ found th a t lii O lab elled g-hydroxyphenyl^ pyruvic acid (gx) was converted to lignin in sugar cane* As sugar cane also contains tyrase the following sequence of reactitxid has been postulated by Welsh (Fig* V )*

g-hydroxyphenylpyruvic s

4 L>tyrosino (Pig.V )

i trana-D-coimarlo acid

m m m

I t seems, lik e ly , th ere fw e, th a t toe 0^-G^ compounds are the most likely precursors of secondary phenolics substances in plants* - 18 -

FURTHER STUDIES WITH PLANT DEUtfgNASES

In view of the relatively high concentrations of tyrosine (%) and 3,U-dihydroxyphenylalanine (^ ) in Vicia faba tis s u e s , i t was of in te re st to examine th is plant fo r the 29 corresponding deaminauses • Neish had shown that tyrase was absent in the legumes, pea (Pisum sativum), lupin (Dupinus albus) and clover (Helilotus alba), but had not examined the broad bean (Vicia faba) and so preliminary* experiments were carried out to see if a tyrosine deaminase could be detected in this plant#

Bean acetone powder was incubated with ^tyrosine and the ether extract of toe acidified digest was found to contain a compound which was chromatographically and electrophoretically* id e n tic a l to c is -p-coumaric acid and d iffered on chromatograms from trans-p-coumaric acid (Table I ).

The of the enzyme-synthesised compound (measured in dilute alkali) was 290 mj|^ (i,e * the same as for c is -p-coumarlc acid), as compared with 333 mju^ fo r the tra n s-isomer (Fig «Y! ), The reason lAy the cis-isomer is formed with the broad- bean tyrosine deaminase, but not with the other plant deaminases examined by Neish, is not at present understood. Our experimental procedures were similar to those of Neish, except toat a Tfis-HCl buffer, instead of a borate buffer, or a mixture of borate and Li_

O -O cO

o - 20 ~ a0 ro 1 I VI I I %##H I I I o? 8 s & I g s I I I I I g- I g-

o #o o # O -*o ■* o o

o o H #o O o ’• w NO ro I % 8 On c VO c o H o o o On CD à CN \0 g

O o H o o o g 8 % & & — 21 —

T>ri&*HCX buffors^ vm used in the msiym digeats* It ia^ of couree, posaibls that the broadf^bean deaminase and NeWi*s tyrase function by slightly different It should also be emphasised again that Neish was unable to find a deaminating mzyme in the three leguminoas species that he examined.

The presence of DOPA has not been noted in many cycles of plants» bat it has been shown to occur in some Viola species aiKi» by chromatography» we have shown that Isatio tinctoria. Alirga reptaas aa%d Toiraya ngristica also contain txao^ of th ia.

Outside th e plant kingdom DOPA i s o f more comnum occurrence» and in both the plant and animal kingdoms is an a intemediate in the biosynthesis of imlanin# It is a particularly interesting oongwund because it is both an amino acid and an ortho- dihydrie phenol.

In the broad-bean» DOPA occurs in unusually h i^ con- oentratlmis and is certainly responsible for the blackening of the tissues Wwn the plant is injured» and at the cmset of s e n ility . I t is also in terestin g to note th a t dopamine ( % ) is responsible for the blackening of bananas (lhasa aaniwtima). - 22 - OH

OH

( )ÔÔ(1 )

llie reasm wlQr such large quantities of JOOPA are presmt in V» faba may be due to either on ateionaally h i^ ra te of eynthesie» or to the inability of the plant to matabolise the amino acid once it has been formed® It was therefore decided to examine suitable plants in an attes^t to discover a DOPA deaminase and» in particular» to see if this mstym was lacking in the broad bean* The deamination of DOPA is» of course» a possible biosynthetic pathway for the formation of oaffeic acid* QRiV)

A prslW nary examination of leaf aoetcme powders from several plant species (tMs consisted of incubating the powders with DOPA and as^eiinlng tha pro*ticta in ether extracts of the digests) suggested WKt a DOPA deaminase existed» and that there was a p a rtic u la rly hig^ a c tiv ity in the dandelion {Taraxacum sj^.)

The product obtaiimd by incubating a dandelion leaf acetome - 23 - ,

powder with DOPA was exa&ined eahaustively» on paper

ohromatogramo and eloctrophoretogranas (Tabla il ) » %n the presence of molÿbdate ions» i t produced a brown coloured» charged complex» a typical reaction of ene-diols» and the preemoe of thia grouping in the molecule was substantiaWL by the observed hig^ €ûa

The presence of a carboxyl grcHip in the product wad a ggssted by the mobility in acetate buffer» (gë 5.2). In a ll systems the product was indistinguishable from authentic trans-oaffeic acid. The ultraf^violet spoctrum of the oosspcmà ma also Idm tical to that of authmitic trans-caffeio acid ami it also shswed the charaeteristio hypecKtomle and bathochromic w - shifts with alkali and borate icsis» respectively (Fig.^l). The latter change in spectrum is» of course» also strong evidence for the eadstenoe of an ene-diol grouping. Gatalytio hydrogenation of the enzymic reaction product resulted in the formation of a compound» wliioh was indistinguieiiable from hydrocaffeio acid (p-3»!$m dihydro:^yph 0wlproprionie acid) m paper chromatograms values in solvmts ^ and B» 0 . 7 9 and 0 .9 3 a respectively)» and eleetrophoretograms value with buffer £» 0.81)» and gave the asm blue-co3u«red azo dye with the diazotlsed gjaitroanilinq/sodium hydroxide spray reagent# — 2l|. — Chromat<«rap^iie and E lectzW iw stle Exaajwatian of BOPA dkwrnlnase predaot able !l(md Qoptim)

value in t Reaction product

ButanolsôtliÆmoliwater (A^- p lain paper 0.71 molybd^ paper U. 0 8 " Ethyl acatatôiac^tio acidtwater 0.6) Tolnme: ac e tic acids water {£) 0.00

10% Aqueous a c e tic acid (O) o U l l Phenol» water (H) 0.3U

2% iquoous acetic acid (J) 0,80

Sodium boz^te baff«? pH 20*0 {J^ 1.16 Ammonium molybdate buffer pH 5*2 (B) 0,98“

Soditsa acetate pH 5.2 (C) 0 J t 8 Amnmium phosphate pH 7.2 (B) 0.61

K Bro*^ mo2ybdate ooeplex formed -2 5 -

1 H v3 2 r o bJ < J % in w 0 CO & 5 1 s I—I Û 0 111 Û 1 Û o È lU aD O o < o h o (f < #- CO y ui o y 2 o <-I Z (Û % vP Z l Z l oS #—41=1 o m rM

5 i| # I■■ Iiiy—— ■ I ■■ l*~ CK OO K vO lO -J- CO c>J T— • • ±_ • ^ o o O o o o o o o

N0IJ.ayO99Y - 26 - During the course of the mzysm reaction^ it wm so established with Ne wsier » reagent that amcmia was also produced» The enzyme producing caffeic acid ir m WPA in the dandelicm is th sre fw e a tru e deaminase, (Fig.Vi) ) F y vij

OH OH frOH + NM5 ------^ + V J V CH,^-CH CH = CH.COj^H V NH: (XXX) (XXtfV)

Quantitative estimation

The DOPA deaminase was estimated by- measuring the rate of ibrmatixm of caffeic acid. The acetone powder^ and DOPA in buffered solution* was incubated for a suitable period of time a t 2^ , The solutim was thm acidified* extracted wil^ et^ser and tiie extract resolv^ on a paper ohrcmatogrm with butaxiolsethanoli water solvent (solv®at A), (Tho latter procedure was included to remove tra c e In^puritles, idiieh were apparently derived from the acetone powder during the incubation. The caffeic acid spot was then located with 0,V, l i ^ t and measured spectrophotometrlGally at 320m|vL after extracti

C l .

rp O O O - 2 8 - of DOPA deaminase was defined as the amonnt of enzyme ifeich woxald catalyse the formation of ImM caffeic acid in 1 hr* lender the conditions described in the eaperlmental section* The rate of formation under these conditions was linear for the first 20-30 rains* of incubation and then rapidly decreased. Browning of the solutions, even in the presence of nitrogen, may have been largely responsible for the "tailing off" of the rates* Unit measurements were taken from the linear portions of the graphs*

The of maximum activity for the dandelion DOPA I deaminase was shown to be pH 8*8 in Tris-HCl b u ffe r (Pig*V)[l )*

Camoarison of DOPA deaminase activity in different plants

Table HI lists the DOPA deaminase activities of acetone powders prepared from various species of plants* - 29 -

Table%

Souroe Age (days) Yield of aoetwie m ite DOPA powder ($ fre sh w t.) deminsM per g. ae st ne powder

Dæwialloa leaves 1 1 0 .6 i 11

Wheat shoots 7 11.!» 0 -kS Barl^ shoots 6 6 .2 2 03 Bean stem and leaves b o 1 0 .9 0 - Î2 Potato tuber (u i^ e le d ) ? l i i .2 0 -0 0

Dajulelion and barlay (Hordsm ep.) possessed relatively high deaainase activities« lAereas broad-bean gave a low activity and no activity could be detected in potato tuber tissue#

Broadmbean cotyledons, germinated and ungezminatW also contained no detectable DOPA deaminase# In the case of the broad-bean leaf and stem there did not appear to be imidi variation in engym activity with the age of the plants (Table 17) « - 30 -

Table

Source |ygL(

0 10 Bean stem 7 and leaves 0 10 lU O 11 21 , 28 0 -1 2 35 0 10 W 0-12 1*9 0-13

Effect of lohibitw i and Actlvatow on

fable V smzmarieea the effect of various potmti&L enaym aetivatore and W iibltw s on the formation of caffeic acid by DOPA deaminase* - 31 ^

V fa b le -

Reagent o*oi^ <>jàMm)L Bslativa rate (fin al cencQntrstisn-(>##lM ) (% of CKHltrOl) J-o 4w/ul Soiu-Hcrtn- *

Sodium zaolybdate 1*3 Sodium borate 68 pKAAA-y t £~chloro laercuriac&tato 1*7 potassium cyanlda 86 Xocysteine 120

Heish found that with crude barley tyrase prepamticme, cyeteiae produced inectivatlon, Wt the thiol activated purified pre» Derations# g^Chloz^mercurlbemwate Inactivated a ll preparati

With dandelion acetm e powder DOPA deamlnase^mo3ytdate tied a maHced W iibitory effect probaWy due to oon^leac formation with th e mas^m substrate# Borate also produced a decrease in activity, preswWOy fw the same reason# Borate W ifors used by Heiah for determining tyrase activity ccuM there ibre not be used with DOPA deaminase. The deaoinam wooM %>pear to require -SH groups for its activity as it was fxÀ/uUA^L ijdiibltad Iqr jg-**efemwourlae#tgt8 and aotiv«>t«d ty oystaiiHi. 81i#xt iahlMtlc»* «Iso oocurrad with oysolda. - 32 -

In «xmolnalon, it may be said that an œuyaa ompable of deominating DOPA with the fomaition of eaffsio aoid eaiats in sone monoootyledeaoue and dlootyledenous plants* At the prwenb time, it is tot known «bother this mayae plays m JjKxnrtant part in the synttoiKUi of eaffaie acid in the plant, lha apparent substrate for tee «uiaya», DOPA, has mly been rto»rtad in the literature to ocour in a few s^wcies, nad it is difficult to asoertaSs teether it is a rare eocqiouad in the plant kingdom, or « heteer i t is cewBnenly oeourring in mnall quantities teidh, in the past, have escapad deteotion. The presenoe of little , w no, dsteotabls DOPA in a species eonXd man that it was m t formed, or ones formed, was very rapidly met­ abolised, perbag» via DOPA dem inase. The low deaminase activ ity in the broadmbean may well account for tee abundance of DOPA in tele plant, althou^ an atnosnally higjh rate of synthesis of tels ooE^xrand could also be an important factor. Tyrosine also appears to be at a high level in ti» bean and tels could also result from the Inability of the plant to mtabolise DOPA, as tyrosiaa is presuaAbly a direct preooreor of DOPA. Da addition, it is inter­ esting to note teat bean tissues contain only small ancmats e f dnoaaio add derivatives.

Future studies in coonsotlcn site DOPA deaainase should include a survey of the ooeurrenoe of DOPA in plants, and a (pantitative examination of the relationteip betwsen this pheaoUe amino acid, the caffeic acid derivatives

Althou^ it is apparent from the preceding discussion th at a great deal is now known adx)ut the b io a^th etio pathways leading to the fwmation of phanolics, virtually no infonmtimi is available in the literature regarding the translocatlcn of these ocn^pounds and their direct precursors. The translocation of phenols has now be^ eamdnsd and is discussed in the second section of this thesis (page 3/^)* - 31*.

- 4* 4 r ' :: '»= /# iv-"

■ :'' % ..

s - .~ . . -■■<# a ...... # # “ “I -, 1#:- m --a * ;# ' . '

' ■ . '-.'\

‘..

*#««

M

# M m - 35 -

TRAHsiocmoN OF m m o w m the (vioia faba)

It is no longer poosiblo to fdcture tra»slooatlon in plants as consisting singly of an toward movmmit of absorbed minerai elements in the xylam and a do%«nmrd movesaont of elaborated nutrients In the phloem. It is now known that organic cos^unda move in the xylsm as well as m the phloem, and some inorganic Ions move in the 5Z,53 phloem# I t i s tru e to say, however, th a t th e phloem content of a plant is mainly cwrganic matter, 90^ being carbohydrates# Zimmrmann in h is review o f tra m s ^ rt in the phloem reports that mvrnmit in opposite directions at any one time and in any one chatmel has never bem found, and that most papers describing mvmms^ in <%x)Clte directions can be m^lained in terms of upward movement in the aqylem and re-translocaticm from the leav^Mi through the phloem. I

little work has been reported on the trans- — 36 — location of pheaollos in plants. It has not been es­ tablished whether natural plant phenols can move down thQ phloem from the leaves %here they are biosynthesised, or Aether the complex, high molecular weight i^enols found in the stems and brandies of plants, such as the tannins or lignin, arise by translocation of smaller, non-phenolle molecules, e.g. acetate, which are then converted to 55 #ien o lic confounds in these organs. Hathway has sta te d that pyrogal4el can be translocated intact in Quercua sp.# but little conclusive evidence has been put fo3 the translocation of higher molecular weight i^enollo y / VJ.Î ' compounds.

The broad-bean (Vicia faba) has now been used to investigate this problem further. This plant was chosen as it is easily cultivated and has a relatively siiȍ)le phenolic content.

An examination of th e bean le a f showed tlia t the main phenolics present were rutin (^^iercetin-3-rutinoside, kaempferol glycosides, quercetin and quercetrin) • - 37 - tjfYosine, 3^ k"dih;yüro]Q^ and ssnall mam&3 of iiatdantlfled derivatives of £- caamarlô, oaffeio, ferulic aM sinaoio lOlds. OH

— é XOH —- ^ütu-u

Q|_| II ^gLuCûSJi- -

HO

^lyceSicU \ O-^üjCOSidiL

The stem epid 0rml.s md inner stem tissu e s also eaehlbitcd a eijïiilar plieiiolic pattmm.

To ascertain whether m y ji^axioia oould be traas^ looated around tho plants (sxperimmts were carried out using «foreign^ phonolSj those not found natura3J^ in the broad^besn^ and which oouM be detected aissXly in the plant tissues by ciiroiaatographlc methods* The movement o f these ca:i|>ounds was iiteasured in cm*/3£) min. down the main stem^ and also relative to the wvwwt of - 38 - resarcijQoI (Table VI).

Résulta of easg&timntQ in i^ioh plants were fW» through the out end of the meân vein of m apical leaf# for vaxying periods from 15 Mn# to 2h hr*# showed that a variety of phenolic ccxsp^unds could be transported down the stem of the plant* The rata of m o v m m t appeared to be dependmt on the sise anchor solubility of the molecule Ot Is possible that other metabolio factors could also be involved) « For example# resorcinol# lAm fed through an apical leaf reached aH parts# including the root# of a 30 cm.^hl^ plant within 30 min«# i^ereas quercetin took 10 hr* to move a sim ilar distance* TablesVjarïdyil^hoir that #%enolio glycosides move more rapidly # a n th eir cor- 59 resending phenolic aglycones* In this connection# Roberts has suggested that because flavones and flavmiols# but not the corresponding glycosides# are r-adsorbM strongly by cellulose : that translocatim of tlieso aglyconas can be hindered unless glyooaylation can occur in the 3- position* %ether glycosylation of the flavcmoids wiiich - 39 - were fed tbrou^i the vein of the leaf# occurred at acme stage# is unknom# but both quercetin and kamp- ferol were found throughout the plants as aglyoonee aftw 2b hr# feeding*

In most cases# the low laoXecolar weight foreign pbsnols were partially converted to the cor­ responding glucoaides# and the latter owipowds were found together with the aglyocmes throughout the plants* In the natural state# it is possible that only glyoosidic or other hydro#%illc derivatives are nomally trans- Co-éH- located* Most phenolic aglyccnes have a toxic effect 51 on the plant* these #ytotoxic corai^ounda are usually lipog^ilic in character# cut possess hydrdphilic gro%q>s and are thm?efore surface active* Sndti cctt^unds could interfere with the function of cell vacuoles# by inter­ action with the tonoplast membrane* Addition of further hydrog^iiiic grotqps# s u ^ as glyoosyl,would muHify the toxicity* in the esq^eriioents described# the phenols were fed in excess and therefore the plant would be inc^mble of completely converting the phenols to glucoaides* When small amounts of resorcind were fed# only resorciiiol-3-glncoeide appeared to be translocated* — 1^0 —

TmwlocafelGO doua £roa th* X»av»so e a s a v à st> m moh «lamtr ra t« t b m the pasaaga of g h m o % » Which hod boon Aid ia ih m i^ iha

Two foreign @c«em, ^n m e tM 9 and D-riboae ware also fad ia throe#* the eut stee», and it «no aux^rlelag to find that they eovad more slowly than manor of the phénols that were teated. Mboee had * rat* of 0*h2 relative to resorolnol wad idwmoa* * relative rate of 0.%». the di ferenoe la rate of the tno engare wee possibly due to the greeter solubility of D «rlboe* in aoueoue solutions*

During the feeding experiemte with ^rlbao# a ra^eieg sugar (X) with a low mobility on paper ohraaatograes was famed In aanll maw&s. It had A values similar to tboee of pentose disac

B) %W*oh also gave a pink colour with Be

It seemed probable at flcat that (X) was a d i- saccharide formed from the l^rihoee* ish^riments with th e broad»bew p lan ts o f various ages showed th a t (X) was only produced by plants from 3 to 12 weeks old*

K lectroj^oretio examin itim showed th a t coBipouod (X) was non-acidlo (eero mobility in acetate bu fsr# pH 5*2 (£}}# and that it had a higher mobility in borate buffer# pH iDeO (^) than aylobiose (It-o-p-g-xylaiyranoi^l- D-xyloae) and therefore probably possessed mere Mjacent hydroxyl ^Eoupa# capable of oos^ploodng with the borate# than xylobioee* (X) also gave a positive reaction with triphenyltetrasolim chloride (spray reag^ot jH)# and therefore was not a 142 linked disacch ride# and it gave i blue colour with atiiline-dipheiylamine (spray reagent ^# Wild) is dm racteristic of a If k linked disacoh irlde# - - ^•Glacosldasd had no effeot on the imknom eugar* In- miffloiont material was available for further étudiés K but these preliminary results suggested that (X) was a 1-^2* linked pentose dissechsride, the linkage possibly having an a*oon£tgaration» and Aram the eleetxopboretie rate of movaaent a t le ast one mmoaaoidiaFide resi

The direct conversion of a nonosaooharids to an oligoaawAaride, if sadi is t6e case. Is unusual in intact plant tiasuaa, and warrants furtiisr inveetigatlcas.

Another technique for investigating the movaaant of phenols in plaits has also been used. The foreign phenol — /P mts dissolved in Tween UO (Esp.Ml)^and a droplet of th is solution placed on the leaf epidermis and allowed to diffuse into the plant* dare again the phenol, and the correspond­ ing glttooslds (in the case of resorcinol, oate

In 1957 Mlttler published results he obtained utilising a cce^letely new teeliniqoe Ibr agcamining the pnlom ew tent of plants# I t has been long aceei^ted Uiat an azAid# during feeding# inserts its stylet specifically into the pbJbom sieve tubes of the host plant# Mlttler devised a method for cutting the stylet away tram the feeding a^phid# leaving i t embedded in the sieve tube# The sieve-tttbe sap of a stem is imdw considerable turgor pressure# wtiich forces the s# ^ the isolated stylet and i t can be made to exude for many hours from the cut mad# The sap can be collected in a capHlaiy tube# the exud&tiw (tranalocation) rate measured and the contents of the sieve tubes examined qualitatively and quantitatively# %e advantage of this technique is that the exudate comes only from the phloem and not from the gAiloem plus surrounding cells. %Ath other methods for exesdnind phloem contents# such as making incisions in the vascular tissue# contamination from surrounding tiss%m is usually inevitable# - 14.6 -

M lttler worked with YulberolaGhnus eallgmee. a large aphid fnm wtiich It is relatively easy to out the stylat. Iti the preswt study# esQDeriiaents were carried out using Tubearolaclmus salîmes feeding on Salix daphnoides. in an a tte s t to discover phmolic ecmipounds ia #ie phloem of this plant* fhaso ex^rimmits had to be curtailed as c a ll sap could not be induced to exude from Urn cut sty le ts. ^urWaer iavestii^atioa into the method# siriowed that Mittlmr*# results were a eulminati^ of many mrnth*# work, and the number of times th at the sty le ts had suocess* fu lly exuded sap were relativ ely few# due possibly to the varying turgor pressures in the willow stems at different periods of the year*

A second approach to this project was an exam- ination of the hon^dew of Iiiberola^toia salîmes feeding on Salix daphaoidea (Bsqp^W) p # e r chromatograms# the idea being that any phenols appearing in the honeydew would probably have been derived tram the willow phloem* m&molie compounds were detected in the honcydew using Spray A and «N» ultraviolet li^ t# but different ooUactions of hmeydew from the same stems# appeared to contain differœ t jgkwnolic contents* - U7 - The honusydew was next compared with the le a f and bazic contents of Salix dap^snoides (£xp<^ ) and althou^ there was some similarity in the ^moiXa present in the bark# leaf and honaydew# results were not very satisfaotozy as the phenols ccmsaon to all three extracts were not easily pcrxoüixt^ identifiable but tyrosine aad^cinoamlc acid derivatives did appear to be present (Table^) #

As many of the phenolics in the broad-bean had bean previously identified# aphids f«edi% on this plant were next examined for #ienolics* Celonids of Microsiphum piei were cultivated on a mW)er of broad-bean plants, ih is species of a#ild was too small to ^ ly the cut stylet tech­ nique but large quantities of hcmsydew were collected and examined on paper chromatograms for phenolic ixuitent. When feeding on the bemi leaves the insects excreted tm to the lower leaves and th is could be easily collected and e x ^ ijied (2aqi.Xyii) chrotmtograMphioally*

In this way# i t was shown that the honoydew did poooùo^ contain phenols tbat were present in the host plant (e#a.

POFA and flavonoids w o t o identified) and# prssuW>ly these coa^pounds originated from the leaf phloem and ware therefore translocatdile materials. - U8 -

Extracts of whole feeding aphids m m also examined m paper chrc*mtogram (&sp.1^) mà ompared with the homydew which had been ’^ntiliced” from insecte which had bean feeding on tlta same item end methanolic oxtraots of the stem# These experiments showed th at Üie haxmydm and stem and insect body extracts a l l oontaiwd similar substances including DOPA and flavonoids (Tabled}).

Colonies of Kicrosiohim p iei were cultivated on the stei# of the beans# and tho plants were then fed wi# foreign phenols throng the main veins of apical leavm (Exp%)l)ll ) # 13% results in Table show that the smaller molecular wei#t# phenols such as quinol# c&tec^ol# resorcinol and forolic add# were fowd in the ^ h id extracts end therefore were presumably presm t in the phloem. I t was noted th at only the free phenols were found in these eoqierimsnts and not the aeoofopanying piienolis glycosides as might be caq^eoted. This may mean that the foreign #mnols are not glucoeylded in the sieve tubes but in other stem tissues# However# another possibility is that piionolies found in the bamydem or in the qphid are modifications of the phlem conatituœts. Hence with Wiols-aphid experiments the possible effect of the #hid itself on these coopounds should always be oonsidered. - U9 -

The amyme activity associated with the iqshid body was th erefo re examined R em its showed th a t buffered aqueous extracts of the insects contained c- and £-gluco8idases# £-galactosidayso and m esterase# capable of hydrolysing chlbrogenic acid* Hienolase (cateoholase) was also presmt whioh oxidised catecâiol and catedbln (Fw}qi«W} end léiidi would preswmbly oxidase other ortho dihydric phenols* It is not kziown where in the aqAid body these enaymes were la ated but i t i s reasonable to assixdie that they are present in l^e gut# and therefore could and probably do modlty phenolics originating from iâic phlAm. Anothmr Intm reeting pocQ ibility is th a t these msymes my be partially derived from the plant itself* In any case# these ensym^ constitute a definite hasard in examining wWle @#iids or Ixmeydew for phloem contents* This disadvantage does not arise with the aphid-head tWmiqne*

These scries of experiments have shown that pheRoUc compounds were present in the phloem of willow and broad-bean m d, therefore# presumably were being transloeated. They have also indicated that foreign phenols fed into the leaves rapidly enter the sieve-tubcs of the stem* The actual medianism of translœation of all organic substances is at present a - go - oontroverslal subject* It seems most probable that natm'aUy occurring ;^enolics are translocated as glycoside derivatives or as derivatives of other hydrophiHe substances# Urn mol# wt* j^dwmole are mrely found in the free state in plant tissues# No phenolic glycosides were found in the aphids whidn had fed on plants th a t ïmà been ti^ated with fored^ phenols# this docs not n ecessarily mean# however# th a t the glycoaldea were not prenant in the phloem# as these could e a sily have bew hydx^lysed ly apliid anaymes. (Them is some evidmoe to suggest that the aphid can hydrolyse arbutin whilst feeding (Table ^)))# The stylet technique wDuld obvi usly be tlae best method for cimoking this if a sni table aphid for the brosd-bami c<^iM be fhimd# Altwnatively# foreign phenol® omM be fed to Salix eg. and the otyiats of Tuiwolaohaus B sllm m used* 5.-3

• SL».

K- ##' ' zmwam^ # uW ik*:#.ÿ^s/t0é -Mz (/:## # # ' '53% - 5% -

D e riv a tiv e

During the past ten yeare boron tr^xhleride fuas proved to bo one of Uae rioet reaotivo sisbataïàC^ knom* It is a strfsng lawia addj and therefore the nmjority of orgaule ùMÎctdoiààl groupe oaa react with this reagent because they mostly eontaiu m oXamemt with a lorn pair of electrons# 15 Oerrard and la^pert have reviewed the reaction of boron triohlwide with all the iwia clashes of organic oci^uoda#

Ciimamio a d d d eriv ativ es are widely d istrib u te d U.Z t)irou#out the plant kingdom# It has bem uhom th a t they are oapabla of inhibiting the growth of parasitic microorganisms ns in certain species and van Swmre showed that tlmy have an W ilbitozy effect on spore germination of %Wat stem rust#

Cinnamic adds have bem found to behave as plant iL-n growth factors. corns plants contain free lydroatycinnamio 29 adds# the majority of these cmpoimds have their carbosyl groups np-S2 asterifiod with glucose or qudnio acid# la addition, the phenolic hydroxyl groups may be methylated, as in ferulic ( XXV ) and - 53 - sin^c add derivatives* In rare instanoes, a ptonollo liydroagrl «,TOttp magr be glucoayXated* E3iaiqpJ.ee of sucti cos^unde are given below* f Ac, x) CUfOH 0 r . V II r/q X C — CH ^ CH ^ -

g. - COünriCXryL gL uccS icÜ t

OH

&LS& O- cuu-d ŸY\ - cou^twoXyL gltLCôSiclX-S c c c ff^ y t cxj^cL gLu.cù$icÜLS Sù/iCLpyL gLuxoSicU. ' V^5-KtmxIUoyyCuaKawAic ^UuosidjL

jO - coLLn4/u"y L g*CuU-iobioSioüi^ ' COpfxûgL g*eu

' — ALSO Cciffcjc cxx^ci 3-^ OW , > j OH A-lso cLO-ct ^ l^~p ~^Lu.cosicLe.

roQ6i/iic OlCjl CH C h - C nO —

Ct t/VI/V O kWy L CpiAUA^ ic a c x c L - 5U -

The reaction of boron trichloride with others is of particular interest because it provides a simple method for the fission of the ether linkage* Alternative reagents for effecting other fission (e.g. l%ndrlodio 'acid^ os a mixture of alkali metal and ammonia ) suffw from the disadvmtage that they are powerful reducing agmits and yields of products are usually low* loron tridilorido has been used successfully by Allm« Bonnerj; Bourne and Sa v ilie fo r th e deaU qrlaticn of sugar derivatives* The reaction conditions using bortm tri- chloride are very mild and hence the yields are good*

With oarbo3cylic esters^ boron trichloride forms a i oomplmc which then deco%x)oes with aoylmoxygon fis s io n (P lg .Q ) or allgrl-o3^g«i fission (fig* @) of the mtmt*

------> E.COCl 4 R»OBClg fig* I S ) 2R.00gE* *BClj ♦ a.COgR* 't (RCOgBCDgO ♦ 3R«ci ♦ m m . Fig.(;^i)

The reaction of boron trichloride with methmylated and esterified eiiuiamie acid derivatives fias now been studied* to see if this reagent would convert all naturally ocmirring cinnamic acid derivatives to the corresponding mono*, di^* or tri» hydrcusycinnasnio adds* Such a technique could be used as an - 55 -

aid to the identificatioa of olnomic acida in plant tiosuee*

I n i t i a l 03eperiment9 were carried out with authentic ferulic ainapic (#)* clxlorogwic 6#^ md 3*k»dimeth(U(ycinnmie acids* %e derivatives* in diehloro» methane* were treated with boron trichloride (EacperiiaentaXXII and XWH ) at »78®* The reaction mixtures were left to stand at rocaa tm ^rature for several hours* before excess borm triohlcjride was removed under reduced pressure* The reaction products were then distilled exhaustively with methanol to destroy any borate complexes* and to remove borate as methyl borate* Finally* the products wore examined on paper (diromatograms and electrophoretograms* The results are given in Table ^ • » $6 »

Raaultg of In itial EaoBrlawrt* «LIA BoM# WWiiWW#/M#Wa*l

£afe?g (aaa Oaptlaa)

Starting values value# Ultraviolet ll^ t M aterial «3A Solvent Solvant Buffer Buffer Buffer Fluorescenoe Fluoreaoence Colour with A B A BC and sfuray A atiimonia

Ferulio 0*86 0.92 o*tô 0*78 0*00 light blue Gre^i-blae Broun add Sinapic 0*80 0.87 0.61 0*82 0.00 Blue Blue Yellow acid Chlorogenio 0*86 0.92 o*kg 0*78 0.00 lig h t blue Grem&»blue Brown add 3*U»dimethox|^ cinnamic acid 0*86 0.92 o M 0.78 0 ^ lig h t blue Ore

The product# gave the same colour r actlcm as the e3q>eeted hydroayclnnaade acid# but behaved dlffermxtly cm paper (dircraatograans and eleetrophoretogram#* Xh particular no strongly addle groupings appeared to be present*

It was then realised that the distlllatl

In order to check this* large scale experiments were carried out reacting boron tridilorlde* followed by methanol* with cinnamic - 5 7 -

acid Itself and with g^ooumarlc* caffele and slnagplo adds# Cinnamic acid (Experlaent XXll) yielded 66$ of a finely crystalline cosqpoiind which analysed as methyl cinnmaate and had the correct inelting point and moleenXar weight# Farther confinaaticm of structare came from comparative infrared and ultraviolet spectroscopy (AK^erimentsXXiii; Fig.^f ^ ) md the preparation of a dibjrcaaoderivativa (^xperiiaeiït Fig# W )#

Ghrcsaatographic and electro#ioretic exazminitlon of the ester showed that it was identical with authentic methyl clnnamate (Table IX ) and the compound &lso gave a positive ester reaction when treated with ferric chloride and hydroxalamine hydrochloride (Bsperimeat ) #

As a final confirmation of structure (xm) was hydrolysed witAn alkali* and cinnamic acid (m) and methyl alcohol* idaatified as hydrolysis products (Experiment W ) »

Sim ilarly* gwcoumaric a d d gave a #dLte crystalline solid in 75$ yield# Comparative examination in the Infrared region showed this product to be idanticil with authentic raathyl-j>-coumaric, tlujs confirming the analytical resu lts# Hydrolysis of (m i with a lk a li gave g»Goumario acid and methyl alcdaol# oJ o o

o o

• o

o C\l X

o

o

o

o

o o o cb - $9 - Caffeio «.eld yielded & orystalline c&sipomd (61% yield) which analysed as methyl caffeate and on hydrolysis gave caffoic acid and methyl alcohol*

With sinaple acid ()^ 3jU#$*trihydro3!ylaiiathyl clnnaiaat© was obtained, as Judged ty melting point and pa^r chromatographic and electrophoretic characterietlce (Table R aad Fig* W ) • The product was fh rth e r id e n tifie d by Gonverel^m to th e more stab le trla c e ta te t# W ) * (ztbsperimmiçfyn) which analysed correctly*

# e n considering the xsachanism by which the reactions described can proceed, there are two fmctional groups ^ id i haw to be examined, i*e* the sethoxyX group and the carboosylic acid grou^*

The suggested mechanism of déméthoxylation is shown in Fig.K #* CH, a yvii 0 L 6 - a 0 , 6 Œ , + a -?e 1 O-H 0 -8 (1 ^

^ HOL + CM3a. 25 - 6 0 -

The reaction of boron triohlcarids with oarboxylie aold was exasnined in 1870 by Guotavson idso stated that the produet from acetio acid and boxen trichloride m a acetyl ohlorids, but later it was ^oun that this was

flCj ^ ;

2- t c R.C [-^Ha 2 6 ( o . COR >3 4 [(fi. CO,)e S]^0 > (C.Co);0]

V

2 (U.Cù^. &ol\ o f v R . c o a ( xxm .) _ ( "J 2 n . c a a te^Oj 2 L(n.co,),e]o ♦ *na - 61 - Flg»T^Hiaho¥S that during the course of the reaction, an anhydride (XLl) is produced and this, in turn, reacts with «7 the boron trichloride to give the acyl chloride*

Thus the principal product expected imaodiateiy after treatmmt of the cinnamic acid derivatives with boron trichloride would be the cori^oponding acyl chloride, and the addition of methanol to tMs would result in methyl ester formation by either of the twj meclianisms suggested in FigW and ^

Fi^ * W

R .coci f

B.CO.OCHj ♦ HCl

B.Oi - CM.CO^ ----- > R.ca • CH.OO^O.MCI,^ ♦ HCl

CHjOH Y R.CH - CU.COOCH^ + HGl + HgO — 62 —

It is not known which of Uiese estérification reactions predcmlnates# Both aim st certainly contribute to the reaction»

Attention was then turned to the application of the method to the identification of naturally occurring daaamie acid derivatives, that is to elassify these ocxnpounds into one of three hydroxycinnamic acid ^families*» An extract of beans of Coffea so,. 92 whicA have a h l^ chlorogenio acid content, was treated with boron trichloride and methanol (BlxpexlmentX)^ and, as expected, methyl eaffeate was produced* The methyl caffeate was further identified by aliuiiine hydrolysis, wladb yielded caffeic acid (Fig*Wl ).

Fig XXI . eloloroqe^vc meTkul ctxfftAc kqt'U(iUiSL/> ajucL pAocLu-c-t

<— SolvieiAJb vjACvUct - 63 -

The flowers of Antlrrhlriim majus contain sm all amounts of _ _ _ % g^ooumarlG caffeic (my), and ferulio acid (W) esters. The boron tidohlorWe/metbanol procedure showed satisfactorily that an extract of these flowers did contain members of the £»coumario acid and oaffeic acid **familiea". (Experiment)^.

Table shows the cmparatlve chromatographic and electro#iorctic behaviour of the naturally occurring cinnamic acids and the hydroxycinn^iates derived from thorn. The best method found for the identification of the latter compounds was p ^ r chromatograi^ using ethyl acetate/acetic add/water solvent (solvent ^ and molyWate im^aregoated paper. This syst^ clearly separates all three esters anà liaa the added advantage that tM caffeic and trlkydroay cincamic acid es.ers move as brown, molybdate ccmipl^Kes which are clearly discernable on the chromatograms. electrophoresis using borate (pEI 10.0) and mo3ybdate (pH $.2) buffers is also a useful procedure for identificatim . On paper the three esters exhibit charact^istio fluorescences with ammonia under ultraviolet H ^ t and also can be located with a diasonium reagent (spray reagent .

Tîtese 33q):irimants have shown that Mron trichloride can be useful for hydrolysis of cinnamic acid esters (difficulty has bom experienced when aold and alkali have been used fw such 94 réactions ) and the removal of methoxfl groups, thus exposing the ^parent** bydroxycinasmde acid in th e form o f a methyl e s te r. - -

There i s , also no doubt th a t borcm tric h lo rid e would i^ydrolyse naturally occurring glycosides sudi as oaffeic acid^3-P-glttcosldo (Æf().

II - 65 - ^11 f 21

t liil: ^ *4 o i» % 8 a 8 <5 Q # # # # # a o o o o o o

8 o o

% » 4o # Ô M n o o o

« o o no I2tt 5 ^ §1 «5 5 # # # # ft IHh o o o o o o

&ft o o o o o

I 5 ft ft o c o o o c

ft ft ft ft o o o o o o

5 3 • 6 6 m

âasti %

- “ - . S'*. , . . ^ ^ .

. %%_«, - ' - ■ b " ^ %» v.^^

. f m - r ->-1^ %£ — 67 —

The tacimlque af paper «leatro^iorwie haa bawB applied In the last tea years with great auocoss to the separabioa 9b of mixtares in varions grw#e of em«ponads taclallng the pbaoolias*

9 t Michl in pabJlsiisd the results of his iimstig^loM into tla» el otrophoresis of o»4ihidrte phenols in borate baffwr, 9i and Coals a and Kraas used borate buffer for the exanlnatloa of phcmollo earbosQ'Ile anWs aid other phonolio derivative#* Sinaeurs 99 and Beibalk wad Sthier have also reported the use of paper aleetro^âiorssis for studies with piHBoliea*

97 Noi'o recently Fridtum baa lanreetlgsted the mavwent of phesoUe annpoaarte in various buffer solutlws* So showed toat« lib s eodins borate, sodism molybdete solution oeb be osod as J2 '■dlMy^dUX^ m Sipmifke macwt fw ^ deW^tlxm mi# ^e%mpLa^^ and Wwt other otmoiwol femturoo eon bo ^ oboieo of oultovlo Wwootml^to oolmtloB»* I t mo ott^irtod thot tho rot# of zKnrammt of plimoMo ^^x#oawo on olm tro# pboretogrmo woo é&pmùm^ oo throw sain footoroi - 68 - (»} Molooular weight / (b) Bflgre* of diosocifltlca of funetlooal i^mpm (c) Ability of tbo pheaol to font oaargod oatyloam# with tbs eoB^oiMnta of idm elootrolyto aolutiea*

A3 yet, howevsr, paper olaotn^Aoroois bas aot bom (tovelopsd to a stage vb&ca it mm be routinely a%*llad to bi|ft noleeaXar w ei^t pheaolle ocaspounds, saeJi «s the flactooolds, sbleb jmaraUy have very lam mobilities because of their relatively 'i# «lass/câierge ratioe. ffKus, mast woiWre bam tooded to use ebromatogr#phy rather than {u^per eleetvophcroels fco* tbo eaaa&aatiom of iiutes gbmols. There are, hmever, soae reports of tho ^ > lio a tio n o f the l a t t e r te«@mlque to tbesa raoleoulesa

Xm order to axbiblt eXeetropbaretle mobility a substeooe KOftt have a net ohurge# If a m leeals eootains m atpul swaber of eoidio sad basio groups it om be made to aequire a net cbargs by aljustiag the pH of tbs «lectrolyte* Xbus mtbecysnlns in tbeir mual salt forme do not migrât# meauurEAla distmeas in neutral buffer aolntioDS, Wt w ill migrate la acidie eolations owing to tbs positive tdtange on the ring o sy ^ atom (\uii).

YUll - 69 - '■ üetaalo# and mvû quit* raplâl^r *t pK 4*6 «oâ )oZ , <03 »L Im m r p^i

of flwwRoliî» htt» Rim bwa a#iw #d %y 104 ttsltte borate baff«rs irfLth cnrceaat deoaitl** *f a*1-1*6 mà/mê

Qnâer the** ecttd.lti.wm flavonea f XUv ). iXacuoBel* (%LV^ a n d glyeostdtc derlvm tlw # of time# me^ioaaàB oom la relK tlott te

ÜM number of flds-bydrosyl groupe praew it la tbu a% *r aoie^* and

of «ae-diol gmapa «ttatdisd to th* arcssatlc rka&t»

Both bgdMMgrl arrattgœ am ta «wq>l*x alth borate, giml«K m etA lvaiy charged apedea* ___ XuV X lV

II 0 0

If KO mwA gfouplas la preeent, tnlgration le segllglble cad 80 the method le aot a^tltoable to e ll ooaptniida in 'Wteea olaaeae*

high Molecular «eight phenolic cwqmuads s till eaonst be osofolly examiaad by hli0 voltage elsetm phoresis owing to low rates of m vm m t» k method abareby the hii#& molwular weight ctaapounds cm be made to move relatively quietly an ^gmr electro- phoretOjgrcBS, by coupling with (U.a8oti#ed earbomylttad and attlphopatad addî: (Tmles X and ^ ) has been invmtigàtad* 'fbe tec^iigue previdm a useful additional mihad for the aaalyele of mistore* of thme #noela* - 70 -

fkxam^ ^m m l0 ewgplW Mltb ÿ^m dm bm&oic mdLd# g lv lo g m&o %dLt# diamooiated cmrboaqrl grouyp»* ?er wltb *rb&tlA 6W (^LVf) la foe^ed»

COiH

XLVI

Such ocotpouai* would be expacted. to here « greater *lectropk»retle mobility thea the free ÿaamls owing to th e ÿmamcrn of the earbemyl groups» Prelimimur ascpsriBente oarriml eat on low nol» wb* fhioole ehaaeo that this was so, md that the am dyes hJ.gr^ato4 s^ypraxixia&ely tw iee as te r as th e perim t itS (henele in borate tantrer*

Suhaequeot exaetiaatioasf higher nolucuier weight empouade gave sW laf résulté* 'fhue, quero#tln (l[l) end hem^erol {^>} which differ strueturally hgr W y one ^ydroayl groop, on position - 71 -

3* can be eeperateâ M tiereeterU y me the a»o ntjnt àeplTmtivee*

% e St99 pheeole ere d iffle u lt te ee;*rm*e eXeerly*

vaîae in sorate W fw Phenol Ooleop c t Nmeme ihcwnl W ameme-eee- -eee-pHbWbexyWee g-eerboayW# la elk eli teâïïttXeUa Maavn 0 » % 8 .6 g irbatiïs med 0*22 O.SO OatwMm crange 0.6S 0.36 Keee^fepel araase 0.12 8.23 tu teo lia lisd o a i 0 . ^ b#%thyl ass- emXta Red 0.60 unlMbLllfAnM Red Q.S2 0 .T 3 ROortâmia 'irme# 0.62 0.61 Qttereetta o ra a e » Q,2S 8.32 Syirto«,ia le d 0,v^ G.J& Reeerâlael»#- flaeoedLde Red 0,16 0 .)) Batia Ormge 0.12 0.36 - 72 -

Rnmol OalflW a f WKMMm# «,., ■nJm tB Bornim w r'fe r • ;3iV ------in rnimm Mmwl

â e a e a le tis C erise 0.51 0.90 â rb u tln Med 0 ,2 2 0 .9 1 C ateobl» Omiÿ» 0 .6 5 1.00 Emempferol le llo a 0,12 0 .7 ) # # r e e t la t$l%m 0.15 1.20 S y ria # » Bed 0 .0 5 0 .6 7 P blorldB ia oreage 0 .6 2 0 . #

Diamtiwd «üphaaUe aeld was f»iW Ut it» a b e tte r rt»egaat, ia thet the aao dgee fwmed wd# the ;me*le meved more M ildly bium la the eeee o f dimotseecl g-wlae» benmie aeidU W # mss premwebly dm te Mm feet tbu^ milg&mle ««id* «re stroager msXàm then the ewreepaadiag (mrboaylle seiôe.

TQa eodlem aeatate W Ter {pÊ 5»8) owiqç^eîa peuseesiog •Mily pbttnelie ®mqie are ioei^fieiwl.ly imiead ta ellaw #leetp@#omtle migretlm, bat these titevim. *o W dltianel eexhcuqrl gemip move rapidly tewarde the eaode.

In view of tble, the wo dyes feraied edth diwetleed - 73 - g-m ine twcuKtle «eid ww# «ocaRinwt in W a Im ffsr, awl,aa « fo o ted , tiMgr wne ncWla, «tterem th# parmt #*w4« m m m%, (tabl* W ).

XII

Mwmel Aae # e is Pbasal is AmW# aoetata boflbr (g) buffer

GWraetia o ao 0 ^ Kaeapferel aOb €d30 fMitecbin 0,08 OdX) Arlmtla 0,23 0.00 lateoU a 0.12 0.00 Rileridsiii 0.31 0,00

7tM aao dys dartm^waa eaa be pmpamd fzen n s fj (Hwli «peeWm# of the pbamol tgr tbo oom ol liiw o oawplliig poaotlaa, follm W fcy piper ^hrcaatognqsbie pw dfiaatioft* fb# latter effeetlm A y renevas em eee reagent and eelcoreâ intorfw iag

«abatanoee* The pweadur* @m also be aaed ia w m jm etiaa with pper ehrcaaategrepl^» fbaa, aa W m em pbeaol aagr be «aonlnoA

OB a ehi^sahp^nea, togetbar w ith ateW lerâ e mpwiada, the paper apnyeâ

M itb cHeaotlsad g-aW wAaM Dlo aaid (or dtaaatlted oalghanilte asid) a n d a tm tetive idantifleatiaa aedto* The eelaw ed opeta

o l u t a d from tb# eiv^stogras sad iwMHMirtBad ty

•leetroptiorM ia, m d the ideatltieatioB ecnfinasd. A»

t h e MO d y e i» etrw igly eoleared e t bi#^b pH vmloee the progreee

of the electropiioretle adgretlw erne be abserved in borate bttffw (pH 10.0).

3.'

% - TS

# ’:/, ‘"'r;?*..: . . :. Tk gÿïÿ., - - \ ;^: / # - .- . . .? .-K--^'^ :u.': ; V-»; &

, ÏÉ 7i;> . : :. :ï?^' 0 '-M 0 0 ^y ^ # * % # i V- . '•: - It / : % a • ^ a*, ( r '■^'.i.A !, -• ' •■•...

fW :# a # # «

:' t % '% # » Vfr-- - 76 -

cnrc

P<^P#r (Anm atog^rapihy m w o arriW qvl% u s i n g gW#^ 1 md Uoo 3 pap$%# win# tW deseeMlng technique* m# the fbllwing solvent s|^tsw | ell prc^rtlow glym are hy volns* except %h%re steted* lOés A Bat«a-l-ol| othanol, water 107 2 St^^l acetate, a c e tic a d d , w ater (5t2t2) )0S c Ethyl %mteW* j^^ridiw* waWr (£ 3X12 or^nie #mse) lop £ ecetie acM* Ww (2ilil)

‘ 341 E jkeetXe enld* cone* %tUre€&klcrl@ e^ld* w W r (30t3iID) 3f> F Tclnme* ecetie edd* w ter (ii*l» i) ic9 10$ w^tie edd tar . 31c H Flujnol, watsr (bottom layer)

^ fÿridine, acetic acid, water ( I t 2 t2 ) lof J 2% ae siic acid SÜ £ Sctauol, pyridifia, water (6«60) distance travelled bgr carbohydrate \ "digtance travelled by glucose

Holybdate pa^r m» obtained by "dipping^ whatmm Ho* 3 paper in laiffer B solutica and aUcKing it to dry in air, befnr* spotting# - 77 - 3 M W i'-

ioy A Diftttotiswl ealntiaa mâ W**»dla* êgrdfaadito (m m ola)

£ »»A«l#lanlao bmsoie wSA and a-oodiaa bydroadd# (îfeatwla) *

% Trlgha^ltotm m liam toXorldle (S i m thaaolio salatioo) 117 " (Sledtoelag aWgeameohaplda* @c*w then l->2 liakto) - 78 - Patwjr JËletctooohorasta

f‘spar elaebroptionrtla mwWi&lw of cocpeunda was oazTlad oat »stttg lAwWaa No* 1 wd io* 3 ÿopor for 3j aia* a t Ü5 v/m» %# boffaro omod warat

11) k 0,2 Hkaodia» borate lO.O) B 8.1 m 10**^ ^arm oalm aoljbtîata

••D 0.3$ M-Irin-acX t* (j« 8.8). X 0.2 K-iMaBoala» ijhosphoto (jK Î.2 )

The w * i l l ti e 8 of pbmola wsra emweooad no %. iWooe.

^ _ diatonoe momd t y pbm ol diatame aoved *qr salioylio aeSâ

The ttohilltles of cazWiydrate# «ore exaroMed aa %XiQ valaao. dietonoe moved, ty earbohydrato / ’^LU uLU dlôt

U - 79 -

Iftfrarod aaaaw rw at»

AU ImfrmrW meaawmmtBwre in anj@l «nllm wLih am ïafraoaml 3peotrojBiotQrMt«r (.%gd*l 137} •

K ltgavA jlat asaaiwgBMWfea

A ll naasapsmsBta in the nltrm vlclm t ragton mar* oarrlad out w ith a ?*nloam S.F*ScX> la ailio a galls (1 em.)«

- 81 -

IProjjBr 'tloa of Aeatono Powder

plant mwAwlal we «ocarstod wWx aoGtoan a t «20* In m wrixig KLmckM"# Tte imultSng pawdmni

V ' ' A w ra filtaro d o ff in « Boebnw IbntMl and wabod flva tim e with more aoetom at . Last tsasw «^.ooetaaa w ra

raoow d antiw w norn and the .)@wd*w w va ataod in dasieaatoni

. ,o a t It •

Ü i^aassg&lsa sL

Iha aaeAam powiar (ou 0«S g«} wa miwd mito about 300 time# it# w l# )t of

ï^ r ia m A ï mmmtirnjgf wlto .toetong. PW*p

A (MBtpla 0£ acetoo» powSar solation (Es^miasoi H } 0.2 g.) «as Wmbstod at 25* with 3,0^m »4iCl baffsr (20 ml.# pH 8 * 8 ) ecntalaiag 0.1% IpSÿrosiae or 0.2% Oi|pOO?A. After 3 hr. the mixture was dHatsd with water (10 *1.), fU tsnd and ttw pB of ths filtrate sdjmted to 6-7 with S^r^l. %# solution was then extracted with ertoer (50 ml.) and the ether «attract evirated to dryosss on a rotary sngx>rat«r. %s residue was finally/ dissolved in ether (1 ml.) fear (Araast<%ra;Aio examinatlca.

The S3FA dsamlnase solmtlaa (prag)sred ty extrosting oppmx. 0.5 g. aoetoos pother w ith 5o ml. o.(g %.frla-8Cl huffer, pH 8 .8 (see 8%. ^ )), was incubated a t 25* with 0.05 g»fris4Igl (So ml.* pH 8,8) oantaloiog 0.2% hl^iFA. At intervals of 10, 20, 8o, 120 and l8o min. portions (10 ml.) wore rmaoved fin» the mixture, acidified with ^-HGl (to pH 6-7) and estreoted with ether (2D ml.). The ether extracts were oaneeatrated to dryness and the résidas radissolved in ether (1 al. etoh). %e ethereal aolutlaos ware « ip lie d quantitatively to s ApomatoGpm *Aioh was developed with Solvent g - 83 - ior 16 hr* The was oIIomkI to dry at room tttaparetun •od then «ownLosd undter altreviolet li# t to dctoet to# ettffnitt aicid spoto. %# "strip#* #f papar omtolBiog to# m id m m «at ont, eiatto wito (08% v/vi 5 ml.) and to# tonerbaae# of «mah «:>luiiom measured at 32i

A ^ayh ef imtoiteetlon tie * agalnet «bswtonee was plotted far eamh aootooa powcUnr (flg.ljf ) sad unite ef astivltr par g. ef pauder ealauliAad from toe linear portiam of ssto grag&.

S.B. A# m it ef Mtlvlty to doflmad #e toe amomt @f «nayne feraiag ImN ef «affale «old to1 iir. under Una steadard eoedltioas daeartbad Above. o oo

\P

- O

le- O

co oJ Ô Ô o M*" o?g NOiidyoSQV — 8^ -

Experiment Y

Conversion o f acid t o o:^«>|?^ooiimaric a o ld

A sample of g^coiaaaric acid (0.10 g.) was dissolved in a slight excess of sodium hydroxide (O.S ^ • This solution was irra d ia te d fo r 2^ h r. hy a 100 366 Hg# source^ shining directly on the surface of the solution from a distance of 3 in . After irradiation^ an excess of H«hydrochlorie add was added and the product extracted hy shaking with ether (30 ml.), fhe ether extract was evaporated under vacuum and the residue rectyBtallised from toluene (m.p, 131^) # The saoule was shown to be free frcm the trans-isomer by examination of the ultraviolet spectrum.

Experiment Vj

D etection of Ammonia from DOPA Deam^jmp@e

Ammonia was detected in the DOPA deaminase digests by the use of Nessler*s reagent. Gkuitrol digests using boiled enzyme solutions produced no anmonia. - 86 -

Expordment Yü

Preparation of Hyürooaffele add

Authentic oaffeic acid (1 g.) in ethanol (20 ml.) was hydrogenated at room temperature using a palladium oxide catalyst (ID mg.) • The product wad purified by paper chromatogra#gr (Solvent ^ and crystallised frcrni ethanol-water, M.pt. li|0® ( m.p. 139®).

Eaperlmen't Y!i*

Oaffeic acdd obtained by the action of dandelion DOPA deaminase on DOPA,was hydrogenated under sim ilar conditions and the product examined directly on paper chrcaaatogrsps (Solvents A and B) against the hydrocaffeic acid prepared in Experiment % . - 87 -

Experiment S

Iso la tio n o f 3>U">dihydroxyphqgylaXanine (DOPA) from Broad-bean leaves

Streaks of leaf extract (in aqueous methanol 8o? v/v) were made on Whatman 3MM papers and the chromatograms developed with solvent S (DOPA has an value of 0*5 in this solvent and is easily separated from the faster running flavortoids and cinnamic acid derivatives in the extract) • The DOPA bands eluted from the papers with water and the eluate concentrated to produce a crystalline compound (m.p. 260^ with decon^sition).

The ultraviolet spectrum (measured in aqueous solution) o f th e extracted DOPA showed absorption maxima a t 230v^|tc, 280 and 310m|w., compared with 230n ^ 2 6 2 and 3l 5»4 ^for authentic DOPA# The infrared spectra of the extracted confound and an authentic specimen of DOPA were identical^ and the two specimens also GO*chromatographed in solvents A (R^ 0.2k) and B (R^ (5.35)• In addition th ^ showed similar electrophoretic mobilities in buffers A and C. - 88 -

Experiment Z

Other Phenollca in the Broad*bean

Fresii leaves (100 g«) were macerated with aoueous methanol (80j8 v/v; 2$0 ml.) • Chlorophyll was removed by continually shaking the extracting with petroleum ether (b.p. k0®*50^). The extract was then filteredand concentrated under reduced pi’essure at kO^.

The extract was examined on paper chromatograms using solvents Wt A, tm 88tE and nS K. These chromatograms showed the presence of queroetin^ quercetrin, rutin and kaen^ferol glycosides. The exact nature of #ie kaes^ferol glycosides was not examined.

The remaining extract was hydrolysed with H*HgSOj^ (3 h r.I 100^). The re su ltin g yellow p re c ip ita te was filtered off* dissolved in aqueous methanol ( 80 ^ v /v ; 5 m l.) and fractionated on sheets of borate impregnated Whatman 3 MM paper using solvent A. SB

One main fraction resulted (E^ 0.78) which was crystallised from aqueous methanol (m.p. 270^ decΠip.). The ultraviolet and infrared spectra of this material was identical with those of authentic kaeiqpferol. - 89 -

A slower moving fra c tio n (E^ 0 . 06) obtained from the paper failed to crystallise* but paper ehromatographio studies in acidic solvents (B and ^ indicated that it was quercetin. Comparative ultraviolet and infrared examination with authentic quercetln confirmed tîiis.

The aqueous* acidic solution remaining after removal of the flavohols was neutralised with barium cazbcnate* concentrated and examined on paper chromatograms in solvent B. Glucose* arabinose and rhamnose were detected.

The extract was also examined on chromatograms for

free cinnamic acids in solvents A*mr B m and F. as The results are tabulated ia îable. Kill

Table

EjP In solvent! Colour with Inference spray A

#*ABFsa m 0.71 0.82 0.00 Brown Oaffeic acid 0 .86 0.93 o.iS Blue Ferulie acid 0 .8 0 0.89 0 .1 1 Bed Blue Sinapic acid 0.88 0.9U ' 0 .0 5 Orange Pur^ple g-Coumarie add ■m ^0 m Experiment ^ Translocation Experiments

Aqueous solations (0.1 w/r) of various phenols and phenolic glycosides vere fed into the cut ends of the main veins of apical leaves of the broad-bean for varying periods of time (10 inin.-3 hr.). This was effected by partially di- secting the main veins from the laminae^ dipping the veins into the phenolic solutions and then cutting them at the apices^ with the veins beneath the surfaces of the solutions (F ig^) #

The plants were then cut into sections (leaves^ cotyledons, root and 1 cm. portions of stem), quickly macerated with aqueous methanol ( 80 ^ w/v) and examined on paper chrcxaatograms using solvents A, E and 0 . Pla^to u>€a-û- vlqjuà . Uxv, aJÀ. CAJiSiJ>

I /b SCiluHoi/v pl\€.UcL - 91 - Experiment )0J Rate Meagurementa

Aqueous so latio n s (0.1% w/v) of various phenols and phenolic glycosides were fed into the leaves of broad«-beaa plants as described in Experiment ^ . The plants were allowed to feed for 1$ min., after Miidh time the stems were cut into 1 cm. sections and Ihe distance the phenolic derivatives had moved, determined by paper ohrcmiatographic examination of aqueous methanolie extracts of the sections using solvent A and spr^ reagent A as described in Experiment . - 92 - Table Y)

Phenol D istance moved down Relative mobility to stem in 10 min*(cm«) Hesorcinol

^Hesorcinol 10 ÙO 1.00 Catechol 9 n 0.90 - Phloroglucinol 9 0.90 Quinol 8 0.60 r Aesculetin lU 1 .1*0 Q uercetin 2 n 0.20 Kaenpferol 3 It 0.30 Oaffeic Acid 7 Mt 0.70 -^ Ferulie Acid 5 lo 0.50 ^ Saligenin 7 \ x 0 .7 0 Arbutin 15 1.50 Aesculin 11* u 1 . 1*0 y Resorcinol p- glucoside 18 \tl% 1 .8 0 i/ Salicin* 11 1.10

K Detected with Spray Reagent D - 93 -

Rate of Upward Movement of Phenolies In

T ablai*

Phenol Distance moved up . Relative movement stem in 1 min.(cm.) to Eesoroinol

Hesorcinol 15 1 .0 0 Catechol 11» 0 .9 3 Phloroglucinol 1 3 .5 0 .9 0 Quinol 12 0.80 A esculetin 1 5 1 .0 0 Quercetin 2 0 .1 3 Kaempferol 2 0 .1 3 Caffeic acid 10 0 .6 7 Ferulie acid 6 0.1*0 S aligenin 1 0 0 .6 7 Arbutin 23 1 .5 3 Aesculin 2 1 1.1*0 Resorcinol glucoside 30 2 .0 0 S a lic in 16 1 .0 7

Rhamnose 3 .8 0 .2 5 Ribose 6 .3 0 *U2 f -

Experiment ^ Introduction of into Hroad#b#w Wawm uging Ttjeen^ltQ

An aqueood ooXatlon of Twaeo-^UO {0 * 02% w/v; $ ml#) MBS added to an aqueouii solution of catWhol ( 0 «1%, w/vj 5 ml#) # A droplet of thie isolation was then placed on the main vein of a broad*bean leaflet and le ft for a|^Hi>oadm ctely 20 min# ihe leaf waa washed in distilled water, dried and cut into sections# Thwm sect ms, together with the stem of the plant, were macerated in aueous methanol ( 80 % w/v) md eamlned m paper chroaatograM in Solvent (See Table W ) - 95 -

Table XiV

Phenol fed Phenols found In leaf and % stem ^

Stem Resorcinol Resorcinol, resorcinol p« glucoside Petiole and lower leaf section Catechol Catechol, catechol p- glucoslde mddle of leaf Saligenin Saligenin, sallqyl p- glucoside Apex of le a f Quinol Quinol, arbutin

CM .

Experiment with Tuberolacfanus aaligaeg

Honqydew from Tuberolachnus salignes feeding on a short branch of Sallx dapbnoidee was coUeeted by allowing it to fall on a glass plate placed beneath l^e branch. The honeydew was dissolved in x-;ater and examined on chromatograms using Solvents ^ B and G, the following materials being observed)

Table XV Solvent value Appearance under Colour with Ü.V. lig h t plus spray A ______

0 . 0k Blue fluorescence 0.32 m Pink 0 .U8 Blue fluorescence 0.55 Red

B 0.12 Blum fluorescence Blue 0.19 Orange 0.25 Bright blue fluorescence 0.32 m o .U o Yellow Yellow 0.62 Bright blue Red fluorescence G 0 .8 $ Orange 0 . 0k Blue fluorescence I - - 97 -

Experiment

WmwlmAnu# «Oteiea with tbw . of W f md bwk from Salix«daonnoldQ 8

Honeydew colleeted firm Tuberolaohnua aaXigoeg as in Exparlwnt was compared chrcmiatogri^Mcally using Solvent m A and m B with methanolie eactraets (80% r/v) ' ^ o f leaves and bark of willow.

Table XVI

SoXvmt Bark Leaf Honeydew

Rf Colour a . Colour Colour Possible rem etiep ...... id e n tity K 0.03 I Blue (W.y.) 0 .0 1 Blue(ü.V.) 0.19 Bl»a(U.V.) 0 .1*6 0.62 Hed with 0.62 Red w ith Spray k Spray A Tyrosine 0.77 Blae(oTv.) 0.81* BlMtU.V.) B n 0.13 Blue(U.V.) ^ 0 .3 31ue

Experiment

Chronatoaraphic Coaparisoa of EacbraiBta of Aphids Feeding on Steaig of Broad-bean# with Stem Content^ and Aphid Honeçydew

I^crosiphum pisi were induced to feed on the stem of a broad-beaa plant for 2k hr. This was effected by stripping all but the apical leaves frcm the plant and then blocking the passage of the aphids to these with cotton wool. (H.plsi normally feeds on leaves of broad-bean).

Approximately half of the aphids were then removed from the stem, anaesthetised with a jet of carbon diox3.de (to prevent excessive movement) and their heads embedded in grease on filter paper. The flap covering the anus of each insect was then stimulated with a needle until a large globule of honeydew had been excreted. This was collected in a capillary tube. The remaining insects were removed from the stem and extracted with a sm all volume of d is tille d water a t room tem perature. Aqueous meth- anolic (80 % w/v) extracts of the bean stem were also prepared, and concentrated.

The three extracts were then compared on chromatograms using solvents ^ B and £ and on paper electrophoretograms with buffer solutions A and C. Both were sprayed with spray reagent

A. The observations are recorded in Table XVjl and , where congparable values of compounds found in the three extracts are given. - 99 -

Table xyn Solvent value of compounds Colour with found in honeydew, Spray A a^hld and stem

0.18 Blue/Brown (DOPA) 0.39 Hed B 0.28 Blue/Brown (DOPA) 0 . 5$ (not honeydew) Red 0.7k Yellow (Flavonoids?) G 0.79 Orange 0.89 Hed •• loo *■ Expeiimept XVjj

Comparison of Phenols in Hicrosiphum p isi Honeydew and Broad* bean leaves

Broad-beaii leav es, covered w ith honeydew from M .pisi., wei-e cut from the plant and washed in a beaker of cold, distilled water, care being taken not to damage the leaves. The resulting solution was filtered and concentrated to a syrup under reduced pressure. An aquai number of "clean" leaves was treated in an identical manner. The two solutions were compared on paper chromatograms using solvents A and G.

Table KVlli

Solvent value of coKÇ)ounds Colour with Spray A found on honeydew covered leaves but not on "clean" leaves

0.03 Brown/Orange 0.29 Red

G 0.15 Brown/Orange o .k l Red - 10 1 - Experiment

Location of Foreign Phenols in Broacl-bean with Ma^ a i p ^l

Aphids were induced to stenwfeed on broad-bean plants (as described in Exp. ), for 18 hrs. The main vein of an apical leaf was then cut and dipped into an aqueous solution (0.1%, w/v) of a foreign phenol. After 1 hr. the aphids were removed, extracted with distilled water, and chromatograms of one extract developed with solvent A and sprayed with Spray reagent A.S

Table XIX

Phenol fed Phenol found in aphid

Quinol Quinol Catechol Catechol Resorcinol Resorcinol Arbutin Quinol, Arbutin Butin Quercetin Ferulie Acid Ferulie Acid Oaffeic acid Caffeic Acid ___ - 302 - Experiment ^ Phenolase Activity In Ma

(a) Qualitative

Two large €^hids were extracted with distilled water (2 ml*) • Catechol (0*01 g.) was dissolved in 0.0$ sodium acetate buffer (pH $.6; 2 ml.) and added to the aphid extract. The reaction mimture was incubated at 30^ for 30 min# A control^ using boiled aphid extract was treated similarly. Appreciable darkening of the digest was observed* After 2h h r. the solution was dark brown but the control digest was only partly discoloured.

A similar experiment was carried out using catechin (3^k;7,3*)l4*~peutahydro3y flavan). This was again oxidised to dark coloured products by the aphid preparation. With tyrosine no darkening resulted.

126 (b) Quantiiative

An aqueous solution of catechol (0«$ 2 ml.) was added to a 0.01 M-sodium acetate buffer (20 ml.) containing aphid extract (1 ml., $ large aphids per ml. buffer solution)* Two control reaction mixtures were also prepared. - 103 -

(i) 0.01 It-sodiim acetate buffer (20 ml.) and K- «B SS catechol solution (2 ml.).

(ii) catechol solution (2 ml.) and boiled aphid I extract (1 ml.) and 0.01 H-sodium9Ct buffer (20 ml.).

The three solutions were incubated at 3$^ and their absorptions measured against 0.01 î^sodima acetate buffer at U20 m at varying periods of time up to 30 min. (FigAl*) # - ioi#--

o CO

LO C \j

O 5NJ

o

< “ - 10 5 - Experiment

Hydrolase Activity in glei

Five large aphids (M.pisl) were extracted with 0.0$ ^sodium acetate buffer (pH $.6;2 ml.). The extract was incubated with the substrate (0.$^ w/v) at 2$^for 36 hr. Control experiments using boiled aphid extracts were also set up. The products were examined on paper chromatogram using solvent B and Spray reagents B and A (with glucose as a standard) •» see Table. XX

Table ^ Substrate (0*j% w/v) Ptydrolysis product Infer^iee Maltose Glucose (trace) i isomaltose Glucose £-glucosidase - present Methyl umg- Glucose glucoside" Raffinose Galactose o-Galactosidase Sucrose (trace) present Cellobiose Methyl ^-D^-glucoside Glucose P«>glucosidase Gentiobiose Glucose present -• lo 6 **• j^ e r iiæ n t

E x p e r im e n t IW was repeated using phenolic glycosides and (iilorogenio acid as substrates.

Table XXI

Substrate Hydrolysis product Inference

Arbutin Glucose Quinol P-glucosidase Salicin Glucose present Saligenin Butin Glucose Quercetin ' Chlorogenic Caffeic acid esterase acid Quinic acidF ] present

K Detected with Spray reagent D Experiment - 107 - Action of Boron Trichloride on Cinnamic Acid

Cirniaiaic acid (h»3 g«) in loethylene dichloride (Ô0 ml.) was cooled to ~76°, and boron trichloride (12 ml.) at the same temperature, was added* After 2 hr. at -78^ the reaction mixture was left to stand overnight at room ten^erature. The excess boron trichloride was removed under reduced pressure and borate removed by repeated distillation with methanol. The reaction mixture was then concentrated to dryness under reduced pressure and d istille d in a Jackson unit at 0.1 mm. Between 68^ and 72^ a pale yellow oil distilled over idiich, on cooling, yielded -sdiite crystals of methyl cinnamate, m.p. 3$*. The product (Æ/) gave a crimson colour (a positive ester test) when treated with hydroxalamine hydrochloride and ferric chloride. (Found* C, 7Uà5lj H, 6.33; 19.09%. Calculated for C, 7U.0$; H, 6.22; -OCH3, 19.13%) 421 lit* m.p. 3^.7 .

Molecular w e i^ t (measured cryoscopically in benaene) was 166 (methyl oinnamate 168). ^ 108 #*

Experiment XXllI cl) Exam-iriation of the infrared spectrum of (jm^)

Results of examination of (XWO in the infrared region

Indicated the presence of a monosubstituted aromatic ring and a

conjugated C=0 bond (peak at 1716 can.**^) with a cis -CaC-conjugated

to a G«0 or -C«C- (peak at I 626 cm.*'^j •

I t was id en tical with authentic methyl cinnamate (Fig.^1) • t) Examination of the u ltraviolet spectrum of

The absorbance of OdOftV) in the ultraviolet region was

measured in alk a li (0.0$ H-sodium hydroxide solu tion). The peak

found at 273*y>|iLwas due to the cinnamoyl grouping.

It was identical with authentic methyl cinnamate (Fig^lV), - 1 0 9 -

!>

8 '-110^

> ;x

g Ll

UJ

o - o CO

o < O -If) CM - I l l -

E3S?©riment XXIV Hydrolysis of Methyl Cinnanate

660% (O.l g.) was dissolved in aqueous sodium hydroxide solution (10^ w/v, 30 ml.) and heated on a boilin-, waterbath for 2 hr. The solution was d istille d and a colourless liquid (W) resulted. *127 The 3,5-dinitroben2;oyl derivative (Xyj) o^ (XU) had m.p. 108^ ( lit. m.p. 1 0 8 ^''.

The white solid remaining after removal of methyl alcohol ()UJ) Mas dissolved in warm water and 2N-sulphuric acid added. Cinnaraic acid (m.p. and mixed m.p. 133^) crystallised out. Experiment Preparation of the Dibromide derivative of

(0.1$ g.) was shaken ifith a saturated solution of bromine in carbon tetrachloride until the reaction mdxture retained its orange colour, the solution vas carefully concentrated under reduced pressure and a white crystalline solid (ÆiïTj 0 .0 8 g.) resulted. It was recrystsillised from petroleum ether (b.p. 60°-80 ^) and dried at $0^ (m.p. 117^). (Found: 38.7$; H, 3.U8; Br, $1.89. Calculated for C, 38.63; H, 3.$U; Br, $1.U3) 122 o lit. m.p. 117 . -iU-

F I G .

4000 3000 2000 1500

1 METHYL ClNNAMATE Ï Compound )Ôÿ « 123

ij^erbient XWI

Examination of the infrared speotrum of (|xZv)

This showed the disappearance of the peak at 1626 cm."^ (of. methylcinnamate spectrum) and indicated that addition across the double bond had taken place. (Fig.^). Experiment XXVI) Action of Boron Trichloride on hydroxy* and methow-cinnamic acid derivatives

Experiment^ was repeated usingi-

£-Coumario acid (L#9 g.) (8o ml.) Caffeic acid (1.02 g.) Sinapic acid (2.13 g.) in methylene chloride j The products were#^ cùjohJÀ^jL ( XXKvO (Vt lotsiAJ-e-cL ( XLVI ) ^ (xKX v;) ) methyl g-coumarate from g-couraaric acid (3.6$ g.^ m.p. 137.5^). (Found! C, 67.2$; H, $.63; 1$.91^. Calculated for C, 67.I1O; H, $.66; »OCH^, 1$.$6$). l i t . m.p. 137 . 6^ 1) methyl caffeate from caffeic acid (0.62 g., m.p. 1$2®)* (Found! G, 61.73; H, $.00; Calculated for C, 61.8$; H, $.19) lit. m.p. 1$2®. 0® 3jUi$-trihydroxy methyl cinnamate from sinapic acid (1.01 g., m.p. 17U^ with decomposition). • Uli -

Experiment XX)#

Acétylation of (%jSi) %u,S - cuA^^^uxxjcU^.

(XftVft) (0 .$ g.) was suspended in ac t ic anhydride ( 5 m l.).

Zinc cliloride (0.0$ g.) was added and the mixture left to stand on a water bath (6o°) for 2 hr. The reaction product if as poured into iced water (100 ml.) and the resulting oil left at 0° overnight.

The oil solidified and was filtered off^ purified by repeated crystallisation from glacial acetic acid and finally crystallised from a chloroform-petroieum ether (b.p. 6o^- 80 ^) mixture

(0.3ii g. m.p. 170** ). lit. m.p. 168 ^.

(Found: C, $7 .Wig H, it.7$

Calculated for ^0^; C, $7.1kg H, k.8o).

Experiment Action of Boron Trichloride on an extract of Coffea sp.

Green beans of Coffea sp. (10 g.) were frozen hard in liquid nitrogen and ground to a fine powder in a ball-m ill.

A methanolic extract of the powder xvas examined by paper chromatography in solvents A, B and G and shown to be rich in chlorogenic acid.

The powder was dried at 60^ under vacuum and then suspended in methylene dichloride and treated with boron trichloride and methanol as in the previous experiments (Experiments XXH_ and W l). The product was evaporated to dryness on a rotary evaporator and then taken up in chloroform for chromatographic examination. The coloured complexes 12$ •*

formed in the reaction^ and tended to streak down paper chromatograms, were insoluble in chloroform#

Action of Boron Trichloride on an Extract of

Flowers (ü ) of Antirrhinum majua were loacerated in aqueous methanol (9o$ w/r; $0 ml.) and the extract evaporated to dryness under vacuum at 6o^.

The resulting solid was suspended in methylene dichloride and treated with boron trichloride as before (Experiment^). As in ExperimentXxiythe resulting methyl esters were dissolved in chloroform for chromatographic examination. . 116 •

Experiment î ^ \

Electrophoreaig of Bmeene#%o*%>»carbo33rlat08

Excess, freshly pr^ared diazotised g^amlno benzoic acid (made 'by the addition of aqueous sodium n itr ite solution ($% w/v in I^HCl; 5 ml.) followed by addition to 30 ml. with d istille d water) was added to an aqueous solution of the phenol#

The dye solution was then streaked on Whatman 3B@^ paper which was developed with Solvent B. The chrcxnatogram was sprsyed with N<*NaOH and the coloured band eluted from the chromatogram with aqueous methanol (8o% v/v| $ m l.). I t was then examined on paper electrophoretograms using Buffers A and C. The paper electrophoretograms were again sprayed with solution to intensify the colour. The parent phenols were examined on the same electrophoretograms. - 1 1 7 -

Experiment MM!

Electrophoresis of Beageneazo^-g^gulphonate

Freshly prepared diazotised sulphanilic acid warn added to a solution of the phenol# The resulting reaction mixture was streaked on IVhatman paper which was developed with Solvent E. The main coloured band was eluted from the chrcHoatogram with aqueous methanol (8o v/v) and concentrated under 1 reduced pressure# It was then examined on paper electrophoretcgramc together with the parent phenol using Buffers k and C. Spraying of the electrophoretograms with N«NaOH solution intensified the i colour. « 1 1 8 -

BEFBaENCæS

. F. "4 - 119 -

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