The structure of the polysaccharide from the alga, Ulva lactuca

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THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSQPHY IXSl THE UNIVERSITY ΠLOIXDOH

by JOYCE WIEIFRED E5HEL BRADING.

M arch, 1950,

ProQuest Number: 10096553

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AWtrmet of Thesis.

J .&# Br&àing.

The styaotore o f the yoXyaaooharlde from g iv e lAsW ea# The p o ljsa e c h a r id e has hoea p u r ifie d ;

a f t e r n i t r i o a e id

o x ld a tio u em ail q u a u titie e o f mueio and eaeeh arlo a c id s have te e n obtained»

The carbohydrate has been m ethylated by

rep eated tre#n en t w ith SesSQd* laOH* the ch lorofci® s o lu b le f r a c t io n bein g fa rth e r m ethylated w ith M el, AggO to a product wi'Ui O m , ÿiX%m

P rio r treatm ent a to g iv e am a o e ty la te d product

or su lp h a te fr e e product* s u ita b le fo r m éthylation* were um dueoesefal•

The p r o p e r tie s o f f u l l y m ethylated amd OIOI3

in s o lu b le fr a c t io n s have been in v e s tig a te d ;

th e former fr a o tio n

was s u b ie s ted to methamoXysis and th e products sepsurated and in v e e ti^ te d ,

Mvlà&me fo r £:3;4-trijaethyl-I»rham ao«e and no

oth er sim ple end group, p a r t ia l ly m ethylated hexose and rhamnose, an anhydro str u c tu r e aM an e s t e r was o b ta in ed .

On autohydrolysia most o f the uronic acid and/or pentose i s hydrolysed w ith about 50^ o f the rhamoose, lea v in g an unstable maoromoleoular residue*

THE STRUCTURE OE THE POLYSACCHARIDE ERCM THE ALGA. ÜLVA LACTUCA.

The author w isiies to ex p ress her s in c e r e thanks to Dr, M* Georg fo r her ad vice and encouragement during the su p e rv isio n o f t h is re se a r c h .

Page. ##

##

{M lpkete e s te r a

##

#*

. #

«•

P r o p e r tie s o f aiih^dro sugars

•• #.

».

iMlplmte e s t e r a lg a l p o ly sa c c h a r id e s

••

*•

».

••

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^ »

7*

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9»

A lg a l p o ly sa c ch a rid es c o o ta la in g u ron ic a c id ».

15 ,

^aggîagj o f the layesU feafcloas on tfae m r a poXyaaQCiiarlàe 16 . E x tra ctio n and p u r if ic a t io n

».

N itr ic acid o x id a tio n M eth ylatioa

,»

»» ».

».

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»,

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£S*

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23 ,

..

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E4,

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30 ,

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34,

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40,

M ethaaolyais end in v e s t ig a t io n o f p rod ucts îîroulc aoid^ fu r fu r a l end m etliyl fu r fu r a l e s tim a tio n s , a u to h y d r o ly sis Experimental work E x tr a c tio n and pur I f 1 c a tio n C it r ic a cid o x id a tio n A c é ty la tio n

»,

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48.

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51 ,

Baryta h y d r o ly sis fo llo w ed by a c é t y la t io n and me thy l a t io n

»,

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. »

M éthylation

».

.»

,,

,,

,,

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53. 54 .

M ethanolysis and sep a ra tio n o f p rod u cts

»,

67 ,

In T C stigatioa o f m ethylated g ly c o s id e s »,

,,

70 .

I n v e s tig a tio n o f e s t e r

,»

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».

Uronic a cid d eterm in ation s »,

,,

..

A u toh yd rolysis and fu r fu r a l e s tim a tio n s H iff” g a io ii_ o J . 5 t^ W tu ra l fe a tu r e s lj-tollofiraph,Y

@4^ ,» .»

..............................

b6 .

39*

i^eaoDOCgiou

'Ihe ia v e s t lg a t io a o f a lg a l p o ly sa cch a rid es has dcT^ped darlag t h i s ceatury and rev ea led e v a r ie ty o f str u c tu r a l ty p e s.

Lihe oth er p o ly sa c ch a rid es, they are

hydrolysed to sisip le sugar u n its by th e a c tio n o f a c id s . Early in v e s tig a to r s id e n t if ie d some o f the sugars p resen t in the hydrolyse te s by tMens o f d e r iv a tiv e s such as osa so u es and during th e same p eriod f a i r l y s p e c i f i c colou r r e a c tio n s w ith phenols were e s ta b lis h e d fo r d e te c tin g h e to s e s , uronie a c id s , e t c . , and methods « er e developed fo r the q u a n tita tiv e estim a tio n o f c e r ta in sugar ty p e s, e . g . by d i s t i l l a t i o n w ith h yd roch loric a c id .

I^^ore p r e c is e

inform ation, p a r tic u la r ly w ith regard to the lin k in g o f the sugars l a the m acron olecole. fo llo w ed w ith the a p p lic a tio n o f m éthylation tech niq ues to p o ly sa c ch a rid es, and the developnent o f p erio d a te o x id a tio n fo r «.-glycol groups and I

chroir^atographio a n a ly s is o f migar® aad augar d e r iv a tiv e s , wii^xln recen t years, provide a new and powerful methods fo r confirm ing the r e s u lt s o f m éth ylation and f o r e lu c id a tin g f in e r d e t a i l s o f s tr u c tu r e . A t the p resen t time the su rvej o f a lg a l p olysacch arid es i s very incof% lete^ the red ( rhodoplx^oeae ) and brown (piiaoophyceae) have been more a z te n s iv e ly in v e s tig a te d than the green ( ohloroph^oeae ) and blue-»green (mjroph^ceae) seaweeds^ but generaX iaatioaa are baaed on sca n t evid en ce.

Ê. Many o f the p o ly sa co h a riâ es are a c id ic , t e e to the p reseace o f s c lp h e r lc a c iâ e s t e r or ca rb o x y lic a cid grou p s.

D iffe r e n t sogar types occu r, the h ezo ses

g lu co se and g a la c to s e , p en to ses and ü^ethyl-ijeatosea, although, so f a r , none o f th ose d isoorered ere e z e lu s ir e to a lg a l carbohydrat e s and s tr u c tu r a l in v e s tig a tio n fo llo w s elo a g sim ila r lines^ and has many d i f f i c u l t i e s in common w ith work in o th er branches o f p olysacch arid e ch em istry. Sulphate e s t e r s , S ilp h a te c e te r a were ^iiown to be p r e se n t in the p o ly sa cch a rid es fr te i many red and brown algae by lieas a t a l,

[l , k ] ,

(q u a lita tiv e t e a t s showed th a t the ash frmn

ig n it io n o f the p o ly sa cch a rid es c o n siste d m ainly o f m etal su lp h ate,

and was n ot removed by d i a l y s i s o f the

p o ly sa cch a rid e.

She o r ig in a l m a te r ia ls gave io n ic

r e a c tio n s fo r m etal but n ot fo r su lp h a te, although th ese were obtained a f t e r h y d r o ly sis w ith a c id o r a l k a l i , "hen the s a l t s o f su lp h ate e s t e r s are ashed a lo n e , only h a lf o f Ihe su lp h a te p rei^ n t i s r e ta in e d in the ash , b ut a l l the sulphate i s re ta in e d i f the substance i s ig n ite d in the presence o f e x c e s s sodium carbonate, or estim a ted in s o lu tio n a f t e r t o t a l h y d r o ly sis , 2 SOS%OMb 2 KCB(^Offe +

ig a it^ ------- > 2

and

1^0)

(and C(g& ï%0)

t e a n t it a t iv e e stim a tio n s o f su lp h ate under th ese

3

.

c ô M it io a s Ÿtfore ca rried o a t aad the au ip h ste la the ash

trcm the m l t s was t&mà to be approxliiiateiy h a lf th a t Obtallied

th e oth er two methods |

t h is was eoofirmed

ladepoBdeatly b j laade Heeu & % [3 ]* Correspoiidjjag a a a ly æ s on the Uiva la c tu o a pôl^ôacoharide [4 ] showed the preseac© o f su lp h ate e s t e r , although aoehrate fig u r e s oould m t be ob taiaed l a e a iH e r lu v e a tig a tio u s owlag to d i f f i c u l t i e s l a p u r if ic a t io n , and owlug to the p reseace o f carbox;?lic a c id grouping which a ls o r e ta in s aodiuEii la the s a l t fona o f the p o ljsa o c h a r id e # It i s noteworthy that g a la c to s e i s alEK>st always found l a p olyeaeoh arid es c o a ta la la g su lp h ate e s te r *

la many c a se s

the su lp h ate has a c t been proved to be attach ed to t h is sugar, but whea the c&rbohydrate c o n s is t s sia ia ly o f g a la c to se , e* g , % sr [d ], Irid aea W n laorb id es [ë], the sulphate mu^t be a tta ch ed to i t and the i s o l a t i o n o f an hydro-galactose d ériva tiv e a { gee n e x t s e c tio n ) c o a fir m th is*

i?'uooidij(i seejKîs to be an ex cep tio n for-8(^v

accounted fo r a s fu c o se , m otels and su lp h ate

is

[&] *

G alactose may be d e te c te d by o x id e tio n w ith n i t r i c a c id , when in so lu b le im clo a cid i s formed and can be f i l t e r e d o f f ,

Phenylmethyl hydrazine i s

separating g a la c to s e from sugar m ixtures [?,

a ls o used fo r 3 ],

llucio

acid has been ob tained from the tJlva p o ly sa cch a rid e, b ut n ot w ith the e a s e , nor in q u a n titie s which in d ic a te th a t ^ k-'l % galtxc^-Sc., 3*3

MTOrtic a ci c4 awci I’SVa x^|o4e.

i\ouj i>e&r\ jte+'exz.f'eii [

4.

g a la o to a e I s a la ^ o r con stifaiB u t, Hy4rol.vai3 o f sm sr galphate e s t e r s . ^ 6 âis«»irory o f uatisual d e r iT a tir e s from su lp h ate e s t e r p olysacch arid e I . e . 3j6-aahydro g a la c to se d e r iv a tiv e s from sgar [@] s t l m l a t e d the la v e s t ig a iio a o f sitiq>le sugar su lp h a te s.

A Sj6-aahydro oot^oaud, methy1 - 3 ;d-

fiuhydro-/3-J)-glucoside. ( i i ) was f i r s t obtained by f le c h e r sad 3aeh [iC ] on a i r l i n e h y d r o ly sis o f m athyl-6-broE sotrla o ety l-^ -i> -slu o o sid e (1 )

CAe

(1 )

(1 1 )

Anhydro oompoaads are a ls o ob tained on h y d r o ly sis o f s u ita b le sugar sul^dwtes

[u].

Ba( m )g GMe H

OH

H

( lll)^ B » th y l-/3 -i> -g sla cto p yraao- i i v) m ethyl-3;6-sn hyd ro-a-D s id e - ô - sulphate galaotop yren osld e a e f i r s t sta g e l a the h y d r o ly sis o f ( i ) i s the l o s s o f Br

w ith the fo m e tio a o f a cartoouium c a t io n .

.hen e s t e r s

5

.

o f sulj^toxic o r sa ip h ou lc aci&o are h^^drolyssd i t la fâfâsm th at t&e oxygen o f the e s t e r l l a k ^ e r t^ a in s atta ch ed to the a cid grong so th a t a oarhoalisa o e tio n la formed

l:m4-oi%B

;ca +

end the s e e h a n im o f anhydro rin g foxsietlon i s iindonbtediy sim ila r in a l l th ese o a s e s . Daring h y d r o ly sis th ere i s very l i t t l ®

teadency fo r

the l o s t -v e group to be rap laeed by a *va io n tta m the h yd rolysin g agent {0H~ from Ba(

or aiB~ from îia œ e i

Sat i t i s r e a d ily rep laced i f a s u ita b ly p la ced n u o le o p h illc group i s a v a ila b le in the auger m o lecu le, such a s - ü from the -OE g ^ u p on %. whltdi can approach % on th e o p p o site aid e £r were o b ta la eâ by ï e r c i r a i aad i'orbes [-Biaonuronie e s t e r ( x x ir ) was ob tain ed a f t e r m etim n olysis w ith Mem. H a . th e extreme r e s is t a n c e to h y d r o ly s is ex clu d es the p o s s i b i l i t y o f fu ren o se r in g fsrm so th at a l g i n i e a c id c o n s is t s o f D-maanuronio a c id r e s id u e s lin k ed l i n e a l l y

,

tliroagh p o s it io n s 1 m d 4

t w ^toufiguration a t

%i s

probably |3 a s tiie p o ly sa cch a rid e shows a la r g e n e g a tiv e r o ta tio n end i s i n agreement w ith X Hay in v e s t lg a t io n e on a lg in a te f i b r e s . A lg ia io a c id h as o n ly r e c e n tly been p a r t i a l l y a o e ty la te d u sin g k etane [8 8 ]. Maanuronle a c id has so fa r o n ly been ob tain ed from a lg a l p o ly sa cch a rid es but both g a la o tu r o n le and g lu o u ro a ie a c id s occur commonly e . g . in p e c t ie m a te r ia l and gums frma l$md p la n t s .

A lg in ie a c id resem b les üie

17

.

o th er p olyuronid es in i t s r e s is t a n c e to Bm % ylatlcn and subsequent h y d r o ly s is . She lOLTa p o ly sa cch a rid e I s thought to co n ta in u ro a ic (o r h eto g lu o o n le) a cid and i s r e s is t a n t to m éth y la tio n .

2h@ tW Llium method h as been tr ie d by

another I n r e s tlg a to r [S9] , lïlth o n t s u e e e s s , probably owing to the p resen ce o f su lp h a te , A lg in ie a c id i s o fte n eceompaaied by p o ly sa cch a rid e eo a ta in in g p en to se, s o s t ooiœonly th e m eth yl-p en tose L -fn oose.

fo ly fn o o s e a ls o o ccu rs wlQi su lp h a te

co n ta in in g m a te r ia l.

She mefâiyl p e n to se ,

L-rhiamJose ( ix r ) h as been siuswa to be

CHI

m . (

x ït

)

p resen t in the TXLra polysaccharide- [4 ] , t h is i s the f i r s t in sta n c e o f the occurrence o f Ahacmose in a lg a l p o ly sa c ch a rid es, although i t i s hnotvn to occur in gmi ara b le and m u cila g es.

18.

sm iA H Y OF im E S T ia A T lo m 01^ T m ÏÏLTÂ POLYSACCHARIDE. m

i n v e s t i g a t i o n o f th e p o l y s a c c h a r i d e m a t e r i a l

o f o t h e r g re e n a l g a e h a s b e e n p u b l i s h e d .

E a r l i e r w ork

o n th e p o l y s a c c h a r id e fro m th e g r e e n a l g a . D iv a L a c tu c a , by M .M .f. P l a n t and B .D . Jo h n so n

[4]

in v o lv e d

i s o l a t i o n o f an a c i d p o l y s a c c h a r i d e a s i t s so d iu m s a lt.

A tte m p te d f r a c t i o n a t i o n by p r e c i p i t a t i o n

i n t o a l c o h o l a t d i f f e r e n t pH v a l u e s d id n o t y i e l d p r o d u c ts Y/hich showed m arked d i f f e r e n c e i a p r o p e r t i e s , e x c e p t th o s e v/hich w ould b e e x p e c te d fro m v a r i a t i o n i n th e p r o p o r t i o n o f a c i d and s a l t fo rm .

The p r e s e n c e

o f s u l p h a t e e s t e r w as e s t a b l i s h e d a lth o u g h q u a n t i t a t i v e a s s a y w as n o t p o s s i b l e , and L -rh n m n o se was i d e n t i f i e d i n th e m e th y la te d a c i d h y d r o l y s i s p r o d u c t s a s th e c r y s t a l l i n e 2 ; 3 ; 4 - t r i m e t h y l - L - r h a m n o s e - a n i l i d e and - p h e a y liiy d r a z o n e . in c o n c lu s iv e , 44 g , CC^.

E v id e n c e f o r u r o n i c a c i d was

--1 0 0 0 g . o f v a rio u s f r a c tio n s y ie ld in g

Q u a n t i t a t i v e e s t i m a t i o n o f m e th y l p e n t o s e ,

by d i s t i l l i n g w ith 1 2 ^ HCl, and w e ig h in g me th y 1 - f u r f u r a l p h l o r o g l u c i d e , a ssu m in g u r o a i c a c i d to b e a b s e n t , i n d i c a t e d t h a t rh ain n o se fo rm ed 2 0 - 2 5 ^ o f th e p o l y s a c c h a r i d e , th e p r o p o r t i o n b e in g g r e a t e r i n m a t e r i a l s o l u b l e i n 50jt> a c i d i f i e d a l c o h o l th a n i n th e i n s o l u b l e f r a c t i o n ; t h e s e e s t i m a t i o n s a l s o i n d i c a t e d t h a t s m a ll q u a n t i t i e s o f p e n to s e a n d /o r u r o n i c a c i d v/ere p r e s e n t i n b o th

19.

f r a c t i o u s b u t n o t p r e s e n t i n s im p le h y d r o l y s i s p r o d u c ts fro m th e s e f r a c t i o n s , S ie i s o l a t i o n o f E

: o - t e tra m e t h y l - j ) - g a l a c t o 3 e -

a n i l i d e fro m m e th y la te d h y d r o l y s i d u c t s by M.M.T Georg ( P l a n t )

[39] , e s t a b l i s h e d th e p r e s e n c e o f

g a l a c t o s e a lth o u g h i t c o u ld n o t b.e o b t a i n e d a s th e p h e n y l- m e th y l- h y d r a z iu e d e r i v a t i v e , P r a c t i c a l l y c o m p le te p u r i f i c a t i o n »

except fo r

th e s e p a r a t i o n o f a s m a ll am ount o f v e r y t e n a c i o u s l y h e l d p r o t e i n , h a s b e e n a c h ie v e d i n r e c a n t w ork b y D,M, H ardy

[40] who h a s d e te r m in e d th e s u l p h a t e a n d

u r o n i c a c i d c o n te n t» e q u i v a l e n t v /e ig h t and i> e r io d a te u p ta k e o f th e p r o d u c t .

C h ro m a to g ra p h ic a n a l y s i s o f

f r a c t i o n s o b t a in e d a f t e r m e th a n o l y s is h a s e s t a b l i s h e d t h e p r e s e n c e o f n -x y lo s e » i s o l a t e d a s c r y s t a l l i n e à i b e n 2y l i d e n e - ] > - x y l o s e - d i m e t h y l a c e t a l | a c i d and s u l p h a t e e s t e r »

a ls o u ro n ic

i n a g re e m e n t w ith o t h e r w o rk ;

and a l s o i n d i c a t e s t h a t g lu c o s e » b u t n o t g a l a c t o s e » p r e s e n t i n th e s o l u b l e m e th a n o ly s is p r o d u c t s . I n th e p r e s e n t w o rk , p u r i f i c a t i o n o f th e I31va c a r b o h y d r a te h a s b e e n c a r r i e d o u t ,

A fte r n i t r i c

a c id o x i d a t i o n m u cic a c i d h a s b e e n o b ta in e d » b u t n o t w ith th e e a s e , n o r i n q u a n t i t i e s i n d i c a t i n g sim p ly bound g a l a c t o s e a s a m a jo r c o n s t i t u e n t ; e v id e n c e f o r s a c c h a r i c a c i d was a l s o o b t a i n e d , i n

is

EC. a g re e m e u t w ith th e c h ro m a to g ra p h ic w ork o f D*M. H a rd y , The p o l y s a c c h a r i d e h a s b e e n m e th y la te d b y r e p e a t e d t r e a tm e n t w ith Meg 5 0 ^ , Ha OH, th e c h lo r o f o r m s o l u b l e f r a c t i o n b e in g f u r t h e r u ie th y la te d w ith M el, AggO t o a p r o d u c t w ith OKle, 3 1 ^ ,

î ? r i o r t r e a t m e n t s to g iv e a n

a c e ty la te d p ro d u c t o r s u lp h a te f r e e p ro d u c t, s u it a b le f o r m é t h y l a t i o n , w ere n o t s u c c e s s f u l ,

The p r o p e r t i e s

o f th e f u l l y m e th y la te d an d CHCI3 i n s o l u b l e f r a c t i o n s h av e b e e n i n v e s t i g a t e d ,

The f o rm e r f r a c t i o n w as

s u b j e c t e d to m e th a n o ly s is an d th e p r o d u c t s s e p a r a t e d and i n v e s t i g a t e d ;

e v id e n c e f o r E ;3 % 4 - tr im e th y lr h a m io s e

and no o t h e r s i n g l e e n d g ro u p , a l s o e v id e n c e f o r p a r t i a l l y m e th y la te d h e x o s e ^ rh a im io s e , a n h y d ro s t r u c t u r e s and a n e s t e r w as o b t a i n e d . On a u t o h y d r o l y s i s b i u r o n i c a c i d seem s to b e p r e f e r e n t i a l l y h y d ro ly s e d , l e a v i n g an u n s t a b l e m a cro m o le c u la r r e s i d u e , From th e s e r e s u l t s s t r u c t u r a l f e a t u r e s o f th e p o l y ­ s a c c h a r id e a r e d i s c u s s e d ( P . 9 4 ) ,

c a lc u la tio n s b a se d on

SO4 c o n t e n t , E q u , w t , , e t c . , a r e s u b j e c t to scaae e r r o r , ow ing to th e c o n ta m in a tin g p r o t e i n , p a r t o f w h ich p e r s i s t s th r o u g h o u t th e m é t h y l a t i o n o f th e p o l y s a c c h a r i d e . I t a l s o seem s p r o b a b le t h a t th e p o ly s a c c h a r id e u se d by E , 0 . Jo h n so n and i n much o f th e p r e s e n t w ork i s som ew hat d i f f e r e n t fro m t h a t u s e d by D.M, H ardy and f o r

21.

th e p r e p a r a t i o n o f th e se c o n d h a t c h o f g l y c o s i d e s , p a r t i c u l a r l y w ith r e s p e c t to p e n to s e c o n t e n t ,

tJ lv a

f r o n d s u s e d i n th e e a r l i e r w ork w ere lo n g and t h i n an d h a d b e e n c o l l e c t e d fro m th e S o u th o f E n g la n d i n e a r l y sum m er; th e m a t e r i a l u s e d l a t e r c o n s i s t e d o f b r o a d f l e s h y f r o n d s and w as o b ta in e d fro m M i l l p o r t , S c o tla n d , i n l a t e sum m er. I t i s q u i t e p o s s i b l e t h a t th e p o l y a a c c h a r i d e w ould v a r y s l i g h t l y w ith th e h a b i t a t an d tim e o f y e a r , p a r t i c u l a r l y i f one w ere a h a p l o id and th e o t h e r a d i p l o i d fo rm .

£,

2 KtmtAffîiQU

Am

gaegam iigs

m

fM

toLYSAce^Rigg.

Muoh o f the p i j ^ a t im s resaved fPtm the fro n d s by «BEtr a c tio n w ith aqaeoaa ace ton e;

tW a c id ic p o ly sacch arid e

was than e x tr a c te d a s sodium s a l t by b o ilin g to e fr o a d s w ito d i la t e sodium carbon ate, p u r ifie d by d ia ly s ia g t l» e x tr a c t i a "Gallophaae" a g a la s t d i l a t e a c e t i c a c id and d i s t i l l e d w a ter, oomrerted to sodium ;% lt, w n c e a tr a te d and p r e c ip ita te d a s a w hite f ib r o u s product by pouring in to a lc o h o l. k r o n e r tie s ere d escrib ed on Î.G8 Th& fr e e a c id form ob tain ed a t the end o f d i a l y s i s was n o t ( x ^ l e t e l y f r e e o f in o rg a n ic m a te r ia l (@ -

a to ,

m ainly Ga88^), b u t ra th e r than fu r to e r prolong d i a l y s i s i t was found m»re e f f e c t i v e to remove the l a s t tr a c e s o f m e t a llic io n s by an io n exchange r e a c tio n , u sin g the r e s in Zeooafb S lS j to e r e a c tio n , ite sia K + ^ Ce

= Be s in ^ Ga ^ S i . e .

the exchange o f m etal fo r hydrogen, i s p r a c t ic a ll y sto ic h io m e tr ic up to 0.05 - O.IO U. {4%

t o i s m s ca r ried

o u t by another experim enter [4o] and a f t e r fu r th e r d i a l y s i s a product o f 0 8^ a sh , pore a c id form, was o b ta in e d . ëractionatigu^

o f th e product was n o t att@sg)ted a t t h i s

p o in t sin c e e a r l i e r a t t e i n t s a t fr a c tio n a tio n [4j had n o t y ie ld e d products which were s i g n if ic a n t ly d if f e r e n t in cerbohydzute c o o ^ s i t i o n .

25

,

NITRIC ACID OXLDATim OP THE POLYSACCHARIDE N i t r i c a c i d o x i d a t i o n o f th e p o l y s a c c h a r i d e w as c a r r i e d o u t s in c e p o l y s a c c h a r i d e s c o n t a in i n g g a l a c t o s e o r g a l a c t u r o a i c a c i d g iv e m u c ic a c i d , and th o s e c o n t a in i n g g lu c o s e o r g l u c u r o n i c a c i d g iv e th e c o r r e s p o n d in g g l u c o s a c c h a r i c a c i d v /h ich may b e i s o l a t e d a s th e p o ta s s iu m h y d ro g e n s a l t . I n i t i a l o x i d a t i o n a t t e m p t s g av e o n ly s l i g h t i n d i c a t i o n o f th e p r e s e n c e o f m u c ic a c i d , o w in g to th e p re se n c e o f in o rg a n ic m a te r ia l , p a r t i c u l a r l y s u l p h a t e , w h ich i s o f s i m i l a r s o l u b i l i t y .

c a lc iu m O x id a tio n

w as r e p e a t e d on w e ll d i a l y s e d a c i d fo rm c a r b o h y d r a te ( a s h ^ 6 ^ ) and a s m a ll q u a n t i t y o f m u c ic a c i d w as o b ta in e d . An o r g a n ic a c i d p o ta s s iu m s a l t w as a l s o i s o l a t e d a n d had s i m i l a r p r o p e r t i e s to iC H s a c c h a ra te .

24,

SSgMllfigMSa

AeetYlfetioa o f th e taoJLYsaccttaglM, I t i s g e a e r a ily fouad th a t whea a p o ly sacch arid e can

.

be f i r s t a c e ty la te d , m éth ylation w ith sim tiltaaeous d e a c s ty la tio n can be e f f e c t e d sm oothly sad e a s i l y .

In

view o f the d i f f i c u l t y o f m eth ylatin g the p o ly sa cch a rid e d ir e c t ly the p o s s i b i l i t y o f preliaiixiary a c é t y la t io n was in v e a tig a te d ,

ïh e a c tio n o f p y rid in e and a c e t ic anhydride

ia the co ld had been attem pted [4 ] j

the main d i f f i c u l t y

was that the carbohydrate formed a herd in tr a c ta b le s o li d in p y rid in e. However, the method o f Carson and Kaelay [42] ^3 used fo r the a c é ty la tio n o f p ectii^ w aa tr ie d a s i t in volved a d if f e r e n t method f o r the p rep aration o f the carbohydrate in p y r i d i n e ,i .e . tlie water i a a sw ollen aqueous g e l was g ra d u a lly rep la ced p a r tly by aceton e and then by p y r id in e Î

but the carbohydrate s t i l l formed hard g ra n u les

in the l a t t e r so lv e n t which were n ot atta ch ed by a c e t i c anhydride, o n ly y ie ld in g a product w ith le a s than one a c e t y l group per two anhydro hexose u n it s a f t e r repeated treatm ent.

Much m a teria l was l o s t in aqueous w ashings, a s

the product was s t i l l water s o lu b le , so th at i t did n o t seeia advantageous to pursue fu r th e r th e p o s s i b i l i t y o f a c é ty la tio n p rio r tc m é th y la tio n , ^ r y t a h y d r o ly sis o f the c o lysooch arid e fo llo w e d b? iÆ /-a c e ty la tio n . (b ) m é th y la tio n : She product ob tained from the p o ly sa cch a rid e a f t e r h y d r o ly sis w ith baryta was subm itted to a c é t y la t io n and

25 méthylation.

.

Varying conditions of "baryta treatment

were investigated;

material could be isolated in

25-30^0 yield containing

-^10^ organically bound

sulphate, and barium sulphate which could not be completely removed,

wuhen acetylated in the manner

desrcibed earlier^ a hard product of obtained.

-^12^ O Ac was

Although there was no marked development of

reducing power on ijydrolysis and the rotation of the product was s till negative, it must have been seriously degraded, for after Diethylating once with 14aO Hand M egSQ ^. there was much loss on dialysis, making i t apparent that pretreatmient vd.th baryta did not yield material suitable for méthylation. Autoh.ydrolysis (P.89) gave an unstable macromolecular residue, unsuitable for méthylation.

26

.

Méthylation of the polysaccharide. Using methyl sulphate and alkali. for the méthylation e^cperimentSj crude sodium salt, precipitated in alcohol, centrifuged, but not dialysed, vias used.

Inorganic and other impurities of small

molecular weight should ultimately be lost in the dialyses involved, any traces of protein not hydrolysed by the alkali would not be expected to become soluble in organic solvents by this treatment. Initially, the addition of cold

NatH and Meg50^

over ten hours, in an atmosphere of nitrogen, was tried; the quantities of reagents being comparable with those employed for the méthylation of agar [27] .

After

seven treatments, v/hen the product had not changed in appearance during three méthylations, the product was worked up and it was possible to extract a small amount of chloroform soluble material (30^ (Me) from the solid product obtained, but it was obvious that there had been much loss of organic material. Possible causes of loss were investigated and the méthylation procedure varied in order to improve the yield of product.

Hydrolysis of unused methyl sulphate

at 100“ was omitted, but the H aO Hwas only partially neutralized so that the mixture remained Just alkaline to the end of dialysis;

although 50^ H gSO ^. was

added v/ith ice cooling it was found that the yield

&7 . of product was ijqproirsû if this stags was ostitted I

satirely, She effect of wamiag the mathylatioa mixtaire with rsâuctiou in the time of méthylation, after the first treatment, was iarestigated, since after one treatment the polysaccharide ^lould be somewhat more stable*

In

coE^aratiTe esperlments, at the higher température of 35-40° slightly lower yield of alcohol soluble material of

higher methozyl content was obtained;

thus warming

speeded up the méthylation procedure without signifleant effect on the product, She methozyl figuré of alcohol soluble product was not increased after six to seven treatments*

She

carboliydrate was generally given seven treeteieats, using the jiTocedure dascribed in the ezperiisental section* In this way a hard yellow white solid laroduot waa obtained, ibLtraction with oraaaio solvents. Extraction witJi CHdjj Et®*t 10* 1 gave material with 2ô;i«*te in 16;^ yield ;

this solvent, when acidified,

extracted further material and acidified alcohol extracted a third amount, leaving a dull green swollen res­ idue.

Shis suggested that methylated acidic material

was pressât, only becoming soluble when liberated from the salt form. In order to obtain as much material as possible for

86

.

further treatosat the erude metl^lated. product was initially extracted with filcohol to which oomceatrated HCi was aûàed m til a faint acid I'eaction on moist Congo paper was obtained, later it was found more satisfactory to extract only the fraction soluble in acidified

for further treatment (see section

on méthylation with U&l^hg^O) ^

Ü M ââx file quanti tj of alcohol soluble material varied as moisture was not rigidly excluded and its presence undoubtedly increased the yield of soluble material, with corre ponding lowering of m ethoxyl figure and increaeë in inorganic contaminants, ïhe yield of chlorofwm soluble product from large scale méthylation was ID^BO^, (Me, 50-£4^. llie organic content of the insoluble fraction was assayed and indicated that the overall recovery of material was ^85^, All these fractions vmre hygroscopic.

Ê9.

After treateeat of methylated^ aloohol % l#le mferial with Mel and %^0 it was possible to extraot ^ 4 ^ of the product with CïîClg, It was aot possible to iacrease this yield sigaificeatly by the use of other solreiits without heary coatsmiiaation by silver, possibly due to oom^Xex formation with fractious containing M or

By

using methylated chloroform soluble material for treatment with lurdie reagents satisfactory yields of product were obtained, the methozyl figure was not increased beyond 31^ during five treatments*

#%ere

was a relatively large increase after one treatment, suggesting estérification, % ihieh was coafimed by #ie production of M eO Hon baryta hydrolysis* kronerties of the methylated uolvsaccharlde.

I^ale yellow powder*

Ash

{lla*Ca,S04, traw o f Agi)

œe* SI}.; corrected for ash* G,

H, 7 , 5 d^>}

H, S*,40 ^-|

corrected for ash

(WeHer and StraussI. =

-54,

(C = 0,4}i in caOLZ)

Ionic sulphate 0,4^ Total sulphate 3.

Vigorous tr^ato«ut with hydrogen chloride ia drj methyl alcohol was accessary to cause «^preoiaWe breaMowa of the methylated polysaccharide to ether soluble lariaterial; treatment with baryta enabled a barium salt to h e separated from glycosides by virtue of its Insolubility i n dry ether, and tiie salt was th m i eoaverted to methyl ester.

The separation was carried out according to

the folio?#lug scheme, average results are given. lïiéthaaolysia product ether Soluble product (40^50^,)

extraction residues

1

A th e r

e % tra c tio n

r --------------------------------------------1

S o lu b le ^ l.v cosides .(

00^1

B arium salts

later

(-10^)

Rotations could aot be observed during methanolysis, but the cimiAge la rotation of -&0 {stai^ting material) to ^^0 (ether soluble product) indicated extensive breakdown. glycosides did not contain nitrogen and were fractionated in vacuo. ogter fractioa was further purified aad investi* gated separately, it did not contain nitrogen, S3ie residues were black aad extremely visoousj

31

,

Hitrogaa aaâ silver were pressât, jau Site glycosides were fractionally d is tille d in vacuo, and the specific rotation, refractive indez and methoxyl content of each fraction v/ere measured.

Samples

were hydrolysed with aqueous acid, losses were heavy where Schiff reactions indicated the presence of $

anhydro structures, and the detection of laevulic acid in one such instance agreed vi th the destruction of anhydro compounds, In order to identify the reducing methylated sugars, partition paper chromatography and the preparation of anilidea was applied, a sample of a major fraction was also fully methylated and investigated.

The results

showed that all fractions were mixtures;

L-rhamnose. isolated

as 2:3:4-trimethyl-L-rhamnose anilide after ælvent extraction, was the only simple end group present, although anhydro structures, produced by hydrolysis of sulphate, may also form end groups in a branched structure. Evidence for glucose rather than galactose was given by chromatography, but the anilides isolated indicated galactose.

Results from chromatography also showed some

difference between the two batches of glycosides which were fractionated, the later material probably containing 2:3- dimethyl-xylo se.

33

.

Investigation of ester. Tkie isolation of ester material confirmed the indications of the presence of -GOOH from méthylation experiiænts and furfural estimations,

The properties

of this fraction suggested a methyl ester of a dimethyl-methyl-hexuronic acid, a crystalline product was obtained, but it was not identical with any established compound.of this type;

another possibility

is that some anhydro hexonic ester is present, "

These results are discussed in the last section.

34,

C.1W , .10 AOIP.

AMw

ÿu:î#o?i*i. sa

t»A

«

sîisa si3g«rs aa4 s s a y r©lste4. esmiîouaas are W lle â « I tb ïïjtïrotjàiortc aeiâ, fiirfmfal aaâ derirstires maj îse ofetaiaaâé i*

iWeœa girs r®rj low yield a of co-iiyâroay-»e tkfifa rfa re i. (S

0^ > c « .œ

mCœ Mesos®

-ao.OSfe.Ç"^ \ff.CHO —> ® ------ m w .h yérœ ty-sm thyl fa r fu r a l

Hexaroaie «oids y ie ld oarWa d iexiâe from tM «ïaimjlia

a siô

wlfch probable farmatiam e f fehea deé^mpeoea fe» farfaaral.

üom 'l&r

â

^.w i-üü.m

îleaar^ie aciâ

-c%

ôh

peutoa»

3,

E eatow a y â e i i f a r f u r s l ( see t i , ï

é.

Methyl p m to m s yield aettiyl ferforei,

farfaral

1% 0^

c iî^

/C a .æ

âàitoyi peatose Û.

0_

c .a tû

e-aethyl fttrfnrel

&-M®to haaoaie a eld a y i e ld e% m A t o r t u r a i ,

ae yield o f ^ h e e n âiaxide ia i , la peaetiealljr

35

.

quantitative and this reaction is used in methods for the estimation of uroaic acids, (see P.86), Determinations on the purified polysacchOTide were carried out by D.M, Hardy [4l] ,

The carbon dioxide

liberated from fully methylated material and from the méthylation residues insoluble in CHClg was determined. Results indicated a loss of

-50^ of uronic acid in

the former and only slight loss in the latter fraction as compared with the starting material, ii'urfural and many of its derivatives give precipitates with solutions of certain reagents, e.g. phloroglueinol, thiobarbituric acid*

The former was considered to be

the most generally satisfactory by Rorris et al* [44] and was used for the estimations on the Ulva polysaccharide. However, the yields of furfural and related products are not quantitative, and the precipitates formed with phloroglueinol are of i l l defined chemical composition; hence rigidly stmidardized conditions for carrying out the reactions have been recommended.

Reliable

empirical tables which give the equivalence between weight of phloroglueinol precipitate and sugar, under standard conditions, were first drawn up by Krober, Tollens et al., using single sugars (e.g. pentoses).

[45] for methyl

Later workers have attempted, by discovering

the causes of the destruction of methyl furfural for

36

,

exanipie, to improve procedm*e, so that results are less dependent on arbitrary conditions. Zullgreen and Tyden |46] recommended the addition of salt to 13,15^ HCI for the distillation, the salt stabilizing the acid concentrations at this level, for losses of methyl furfural are very dependent on acid concentration [4?] which varied during the older procedure of distilling with 1£^ HCI,

Losses due to

oxidation can be avoided by working in nitrogen [47] , Quantitative yields of methyl furfural can be obtained by distilling it off in steam at 11o' from 13.15^ HCI saturated with NaCl [48, 49] , Norris et al* carried out estiniations on various sugars and obtained expressions relating weight of sugar to weight of phloroglueinol precipitate^ P . e.g. i'or anhydro rhainnose^ R . R = 1.0433? i-' 0.0118

r 0.0003

for 0.04 g. to 0.16 g. Rhamnose, But Hiese investigators pointed out that the conversion factors are very dependent on conditions and stressed the necessity for investigators to determine the constants for their own conditions, -r They also investigated mixtures of two sugar types.

37

.

e.g. methyl pentose and iironic acid, such as occur in natural polysaccharides.

In general the course of

the distillation of one component was affected by the presence of the other, so that expressions obtained for single sugars cannot be applied with accuracy*

With

uronic acid as one component, a satisfactory relation could be deduced if the uronic acid was independently estimated by

ew®ktti'pn.

Hezoses increased the weight of

phloroglueinol precipitate owing to the production of small amounts of OH-methyl-furfural. In the present work, the method of îicrris was modified for use with smaller quantities of sugars, using rhamnose, arabinose and galacturonic acid. It was clear that the method of Van der Haar [50] for separating methyl furfural phloroglucide precipitate from furfural phloroglucide by virtue of the solubility of the former in

alcohol at 60" was unreliable (as

stated by Morris) although it was used to give a comparative measure of rhamnose lost and retained in the macromolecule during autohydrolysis, !Ehe appearance and solubility of the j,rvcipi+ t: from the Ulva polysaccharide were very like those from arabinose and galacturonic acid, although the

38

.

proportion of alcohol soluble material was much greater, confirming the presence of methyl pentose* In the earlier work [4] there was no indication of pentose in the methylated hydrolysis products, and the evidence for uronic acid was inconclusive, but this work suggested that uronic acid and/or pentose was present, and was supported by the present méthylation and chromatography studies [40] * Autoh.ydrolysis and furfural estimations. Since there was evidence that the proportion of methyl pentose in alcohol soluble hydrolysis products was greater than that in the original [4] there was the possibility that it could be preferentially hydrolysed from the macromolecule *

Autohydrolysis of

the acid form of the Ulva polysaccharide was carried out and the product dialysed;

the metliyl pentose

and pentose or uroaic acid in dialysable and non dialysable fractions were approximately assayed* I’he results indicated a preferential hydrolysis of uronic acid (or pentose), and that di- and possibly tri-saccharide fragments must have been liberated on autohydrolysis, the uronic acid may be liberated as aldobionic acid as is found, after the hydrolysis of

39

.

various plant gums and mucilages e.g. gum arable [51]. The non-dialysable portion contained sulphate ester.

40

,

SSXgESIMEKgâL BXSRAgj’XOü MiU P m i^ IC A lim Qg' flîE g01.YSACiH>mî0S

t i i # LAazMÙA.

'

Ulra laetaoa was obtained from the Msriae biological Station, Millport. Hsgtoval of oijsiaeata. ïigjaeata were extracted from t&e fronds by covering them with 8S^. acetone in eballoe tanks and allowing them to stand exposed to sunlight for $-4 days, stirring Qceasioaally.

Three treatments yielded pale yellow

coloured fronds which were dried in air. original extraction procedure. Initially the procedure developed by S.D, Johnson [4 ]

waa eaiployed wife slight modification. i.e . 100 g, dry fronds were heated with 8 h 0,5^

sodium carbonate solution,on a boilie^ water bath for Û hours and the extract filtered off through calico. She fronds were further extracted by three treatments with 8 1

PJ>5ji sodium carbonate, involving 30 hours

BK >re heating.

The coshined extracts were concentrated

on a water bath to about IB L, filtered through fine cloth to separata from a sludge, which was mainly inorganic, end farther concentrated to a volume of approximately 600 ml.

The polysaccharide was

precipitated in a Wiite fibrous form by pom'iug the dark coloured, thick solution in e fine stream into

41 5 L, alcohol which waa rapldl;^ stirred j

.

the final

alcohol concentration being 88-^0^# She onljr alteration to this procedure was that concentrated hydrochloric acid was added to the sodim carbonate solution during concentration^ in order to reduce the alkalinity and hence minimise the hydrolysis of sulphate ester groups known to be preaent. The solution was kept alkaline to liteus^ the total volume of concentrated HCI used initially was -GO ml. equivalent to

of the sodium carbonate used*

The product waa kept under alcohol until required. It could be worked up to a fairly whiW solid by centrifuging off &e alcohol^ triturating twice with alcohol and then with ether* This crude polysacc>iaride was found to have a very high inorganic content, ash 40*50^ of dry weight,

A

large part of this inorganic material consisted of sodium chloride and soditmi carbonate, ^Aici) have only slight solubility la alcohol ( 0,3 gl"^ IfeCl,0.2 gL"^ i^a^CÛ5 in 80^ alcohol) but which should be removed by dialysis against water;

Oa ’ and

were also known

to be present* PieJLysis ea^uerimeats for yie aurification of the

polysacchagidiL> Cellophane" casings were used for dialysis*

ahen

dry they are easily damaged by contact with brown paper etc*, and so were stored in a damp atmosphere (over 1 ^

42

.

formalin solution) when not in use. The performance of the casings was first investigated by dialysing a solution containing known amounts of sodium chloride, calcium sulphate, glucose and starch against distilled water.

2he solution

inside the casing and the dialysates were tested for the substances used. Glucose was estimated by hypoiodite titration, the loss involved on concentrating was corrected for by carrying out a comparable concentration of a standard solution;

ions were assayed by suitably

concentrating and diluting until the limits of sensitivity of the following tests were reached^and by comparing v/ith standard solutions. Cl^.

0.8 ml. (2 drops) 3^ AgNOg solution added to 1 ml. Cl^ solution, cloud detectable at 1 ^ 10"®g. d' L“l .

3% ^

0,8 ml. (2 drops) 12^ BaClg solution added to 1 ml, SO ^ solution, cloud detectable at

7 X 1 0 -3 g . SO4 Ca

L-1.

2 drops saturated

(

)^.Fe( C JN )g solution ^1 drop

alcohol added to 1 drop Ca solution, cloud detectable after -f- min. at 5 x 10“^g, Ca”

43

.

Results showed that the iaorganic ions, glucose and a small proportion of the starch (giving a red colour with Ig) dialysed readily and that the remainder of the starch (Ig colour, blue) was retained through prolonged dialysis. A solution of the crude polysaccharide, 8 g. organic material, was dialysed against distilled water, and then against dilute HCI of pH 3 (bromphenyl blue), the pH of the acid form of the polysaccharide [4J.

The loss

of inorganic ions was estimated approximately using the nephelometric tests, P.42, After 2 days Cl

0.4 g.

2 days

6 days

3 treatments

HCI (pH 3)

0.015 g. 0 .0 2 g.

S04 0.69 g.

0 .0 1 g.

Ca O.OS g.

0 .0 0 1 g. 0 .0 0 1 g. 0.01 g.

0 .0 2 g.

0.016 g. 0.016 g. 0.016 g. O.OCA g.

Final yield 6 g. organic material i.e .

-75^.

The loss of 25^ organic material on dialysis is not mainly carbohydrate in nature as Molisch tests on dialysates were only slightly positive.

The steady loss

of sulphate is probably due to the fact that solid G aSO^ is present and is only removed slowly because of its low

44 solubility (0.2 at 18* I.

.

Dialysis against dilute HCi

resioved calcium more rapidly but prolonged exposure to mineral acid was not considered safe for the polysacclxaride* particularly If it contained uronic acid. A product woriced up as sodium salt gare ignition.

asii on

A product wor&ed up as acid, by precipitating

O^SodÎH*^Sai^

a solutionnât pH 3^in oloohol^ gare X I f aah on ignition, but this wns probably partly sodim salt, aad %uld contain sodium chloride as this was not reared by dialysis;

Ca

and Pe

were also present.

Samples of sodium salt purified in this way, ash) were ignited and the sulphate estimated gravlmetri* cally as BaS%. O n ignition without sodium carbonate, ”

with

^

S%

, 13$ a%

liie ash fx‘om sodium salt which had only been dialysed for a short period against distilled water was found to contain £2^ phosphate (by "molybdenum blue** test, estimated colorimetri cally).

In riew of the presence

of caleiina and phosphate it was obviously necessary to dialyse against a solution sufficiently acid to dissolve calcium phosphate.

Dialysis against acetic acid was

tlierefore tried as this would not affect the carbohydrate. The concentrated sodima carbonate extracts were not precipiteted Into alcohol, but the solution wa^ neutralised and dialysed;-

45

.

(1 ) âgalA st ra p id ly flow in g tap water f o r 24 boara; t h is reimved m c h o f the I&aCl aad provided th a t the flo w waa f a s t , Cas% did not p r e c ip ita te in the "eeliopbaiie*’, (2} A gainst flo w in g d i la t e a c e t ic a c id . (S ) A gainst flo w in g d i s t i l l e d w ater. a m # le s o f

a cid form p olysacch arid e s o la tio n

were withdrawn a t t h i s p o in t and could he concentrated at

80* w ith ou t ch a rrin g .

tstie fs

On eahing the dry s o lid ,

o f the a c id could be o b ta in e d .

Id e a lly

t h is product should n ot le a v e eay re sid u e on i g n it io n . fh e $ aa^ was used a s a c r it e r io n o f improvement in p u r ific a tio n a s ( 2 ) and (S) were v a r ie d .

In ( 2 )

^1 0 0 , ^ / 60 and

' 2 5 AeGB were tr ie d and i t was found K/ th a t three days d i a l y s i s a g a in st SO AeCH was n ecessa ry fo r the e o i^ le t e removal o f p hosp h ate.

In {3} the

time o f d i a l y s i s a g a in st d i s t i l l e d water was v a ried frenot three to s ix d ays, ash f ig u r e s showed o n ly s l i g h t improvcæieat a f t e r fo u r days ( see overleaf). , A ssista n ce from M iss S.H.M,Saunders in the experim ental work in v o lv ed in e x tr a c tin g the p o ly sa cch a rid e, e t c . , i s g r a t e f u lly acknowledged.

46

e.q.

^'*^100 3

Ash from sodium s a l t .

D i s t i l l e d Ash from a cid form water d i a ly s is

Batch HAc d i a ly s i s 62

. 260 pptd pH 9

daja 3 days (SC^Ca SZySOi 1 .5 ^ 0 ^

63

% 0 2i 3

69

"!

2 1 .1 4

"

I

"i

5,5)1 (PQ4 - 1;^ o f ash)

i .. -. . . . . . . i

4

"

3

5 6

” ”

"

" " 11

;

; 5 .4 ^ 0 4

5 .4 ^

1

=

of ash) i 1

!

Later batches were dialysed against '25 HAc for 1 day, ^/ôO HAc for tv;o. days and distilled water for 4 days and gare figures generally varying between 5.5 and 6.0ÿb ash in acid form.

Ilhis ash consisted mainly of CaSO^; Ca

was removed by the ion exchange resin "Zeocarb"215 in its acid form and the 3 O 4

by dialysis {D.M, Hardy[4o]U

for precipitation the solution was made alkaline with 2 14. i4ame p rod u ct had been l o s t l a th e aqueous w ash in ga, %he r e s id u e H, a f t e r w ashing w ith p y r id in e end c e n t r if u g in g was a llo w ed to sta n d w ith

10

m l. w a te r, i t

s w e lle d to a s o f t g e l , sue 1 1 lo g s tr o n g ly o f p y r id in e ; on a d d itio n , o f SO e l . p y r id in e w ith s t i r r i n g e more f l o c c u l e n t p r e c i p i t a t e was o b ta in e d ,

% e p y r id in e was

removed by c e n t r if u g in g and th r e e fu r th e r p y r id in e tr e e W a n ts a p p lie d , each w ith 24 hours sta n d in g ;

the

r a th e r hard p ro d u ct was a c e t y la t e d in th e same way a s b e fo r e , i t d id n o t go in t o s o l u t i o n .

A fte r sta n d in g a t

room tem perature f o r 48 h ou rs th e whole v s s poured in t o

.

53 150 m l. 3^ HQl axià woriceâ Acetyl Gomtemt 13^ (

aa before* ^ ëP per aiihyéroW^oae uâiti*

O verall y ie ld 45-50^* o f a c e ty l coiiteat was carried out by s t ir r ia g a kaomi weight o f im te r la l %iith 0*5 m- a lc o h o lic iiaOH fo r

hour a a t 65' l a a stream o f uitrogeu* t it r a t in g

exoesa I%OH w ith o*6

HCl uaing phenol p h th alela in d ica to r

and comparing w ith a blank eaperlmeat, e .g .

g.

dry wt* of material treated

with Bxoess i4cH Blank 1

10

m l. 0#5 a a lc o h o lic Ka®«

10.039 ml* 0.463 ïi H d . 11.075

gEi. oertohjrarete coatalzis f

o ^ *T'ï oÜ^^" ""

=0.1S gm, Oac , BARYTA HÏ0R0LY3XS. A 6)4 a o lu tlo a o f oarbohyârato la a t 70" In aa atmosphere o f a ltr c g e a ,

baryta was healed % fiim s a l t was

p r e c ip ita te d aad a t ao p o in t did a l l the organic matter go in to s o ln tio n , S eatin g time

Bedncing power as m l,

2^

hours

1 . 6 'm l.

5

hours

2 .6

N/

/lo

m l.

Baryta concentration increased to 10^ 22

hours

2

m l.

Oa a c id ific a t io n w ith HjjSOij (to coago) the p r e c ip ita te could be cen trifu gea o ff, lea v in g a dark pungent s m ilin g

-1

.

54 saXutitm.

YleX4, after ooaoantratlag aad j^reaipitatioa

aleohaX,

(iaoXaâiiig aahL

 similar h;ydrol,y8la at 88“ showed a reâaoiîig figure

ÏU

3.5

ml.

10 % after B iz hours which fell to uear

aero oreraight.

Ifehllage aad t^iliwaaoff teste aegatlre.

Ihe soXtttioa was rotieatad w im baryte at 80" m à worked up aa before. recorery of organic material (with 38^ aah). C'^]

*30 (1^ aqueous solution» cloudy).

Baryta hydrolysis at 100" yielded a very viscous degraded product. Acétylation of baryta treated material» aeoordiag to procedure given, yielded a small quantity of hard material

OAe =

1 8 ^.

5 gm. was methylated

with M e^SO^ and llaOH at room

temperature» very little solid separated out on dialysis, fotal yield 30^,. containing Ba8(% ;

M4ŒY1AT1UB #

13^

m u HOLYSAOmAmm.

wly^amhmride m i i m methyl sulphate and

P reim m ary Qinerimntm. Crude sodium salt fom carbohydrate» precipitated into alcohol and centrifuged, was used without further treatment, e*g# drying. Material corresponding to 6.^ gm. dry dialysed organic

DlACfR^M 1.

55. m a te r ia l was s w a lle a to a g e l w ith w ater euid œ t h ^ la t e â a t room tem peratare w ith 8 9 ü m l. SO^ MeOR and 140 m l. MegiO*, u sin g the apparatus shewa i a diagram 1 .

îh e

re a g ea ta were added s lo w ly , through the two tap f u a a e ls , i a te n th p o r tio n s a t h ou rly i n t e r r a is ; a stea d y stream o f n itr o g e n was p assed in t o the f l a s k through the tu h e. A, and the ml%ta r e was s t ir r e d r ig o r o u s ly ,

At th e end o f

ton h o u rs, any m ethyl sulpha te rem aining was h yd rolysed by h e a tin g on a b o ilin g w ater b aih f o r 45 m in u tes, w ith slow s t i r r i n g ,

The carbohydrate darkened oa treatm en t

w ith a l k a l i , more so on h e a tin g . w ith

A fter n e u t r a liz in g

the prod uct was d ia ly s e d a g a in s t d i s t i l l e d

w ater f o r

- 5 d a y s.

ïh e d ia ly s e d s o lu t io n was

co n cen tra ted a t 45° and then rem eth yln ted setreral tim e s, u sin g g r a d u a lly reduced q u a n t it ie s o f r e a g e n ts w ith a d d itio n o f a c e to n e . Second m é th y la tio n , a s f i r s t . % ird to f i f t h m é th y la tio n s , 240 m l. 8(^ lieCM, 100

m l, Keg30^, 8 0 m l. a c e to n e .

3 l i t h end sev en th m é th y la tio n s, 220 m l. 80^ IkcH, 90 m l, Meg9% , 120 m l. a c e to n e , The appeerenoe o f the product and i t s i n s o l u b i l i t y in w ater d id n o t a p p r e c ia b ly a l t e r a f t e r 4 -5 tr e a tm e n ts. Üie f i n a l d i a l y s i s was allow ed to run f o r 17 d ays and oa co n cen tra tin g to d ry n ess 1 ,7 g . o f a l i g h t y e llo w

56. powder was obtained* which was e x tr a c te d th ree tim es w ith b o ilin g ch lo ro fo rm . Yield* Ü.27 gm .;

m e,

Methoxjrl f ig u r e s were estlE ia ted by the sem i-m icro Z e le e l m ethod. F a cto rs causioK l o s s o f rria teria l. M a teria l must hare been l o s t during th e d ia ly s e s * co n cen trâ t io n o f d ia ly j ^ t e s from two m é th y la tio n s showed th a t th ere was a stead y sm all l o s s o f o r g a n ic s^iaterial, and th a t m )st o f the aodima su lp h a te was d ia ly s e d a f t e r 56 hours w ith d i s t i l l e d water* changed h o u rs.

ev ery e ig h t

Hence* e x c e p t f o r p u r if ic a t io n in the l a s t sta g es*

lauch slîo r te r d i a l y s i s tim es were employed ( se e account o f f i n a l method em ployed .) tlhe a c t io n o f c o ld and h o t 30^ teCM was in v e s t ig a t e d , Ji 2^^ s o lu t io n o f the carbohydrate in

lle œ was s t ir r e d

v igorou sly^ in an atsiosphere o f nitrogen^ a t room temperature^ fo r 10 h o u r s , fo llo w e d by 1 hour oa a b o ilin g w ater b a th . â t the tiia es in d ic a te d a liq u o t p o r tio n s were withdrawn and n e u tr a lis e d * and the red u cin g power determ ined by the w i l l s t a t t e r hyixsLolite method a s ïio d ifie d by Baker and H ultou [6 3 ],

^ i s i s n ot a s a cc u r a te a s l a t e r HK>difications

u sin g c a i'e fu lly b u ffe r e d s l i g h t l y a lk a lin e c o n d itio n s e . g . [53] arid was fu r th e r com p licated by the f a c t th a t z' *

io d o fo n a cou ld be d e te c te d a f t e r t i t r a t i o n , however ciianges i a Io d in e uptaice g iv e aa I n d ic a tio n o f the li b e r a t io n o f

s? . red u cin g g ro u p s.

1:

Hours a t 20° in 2%

H |g -

-1 m l. /lO l£ gm 0.1 0 , 3 |4 .0 i . ^ . 4 Hours a t 100" la % m i. %

%gm”^

0

H

H

4 .7 4 .6 4 . 4 | 8 . 0 i l . $ : |1 .4

1 ilO h r . a t 2 .4

8|

8 ,0

2 0 “ ia a ir . 1 4 ,8

K etfiy la tio tt was r e p e a te d , o m ittin g th e h y d r o ly s is o f o n ly about Z5^ o f the 50^ HgS% req u ired f o r com plete n e u t r a lis a t io n was added b e fo r e d i a l y s i s (e x c e p t f o r sev en th lœ th y la tio n ) and l o s s e s were v i s i b l y much l e s s , 1Ï 16

dry prod uct was e x tr a c te d w ith CSClSjEtCHs { l O t l ,

Y ie ld , lô^ i) f o r 8 - 5

h o u rs, n e u t r a liz in g w ith

69. AggOOg fUld e x t r a c tin g w ith MeoH| d r ie d w i #

and tie OH removed.

th e e x t r a c t vsas ühe product

co n ta in ed tr a c e s o f Ba toad A§ s a l t s h u t could he fr e e d froBi th e se by €H|A

1

110 - 115 ' 0 .0 5 5

Intermeit i a t e

Added 5 & 4 B .3

115-

j

0.619

4 5 .S

1 .4 5 6 2 (2 5 °)

5 1 .3

1 .4 6 3 3 (1 8 °)

122 - 130 B ,4

144 - 148

0.199

i.

B.5

158 - 168

0.0 4 5

i

B.6

170 - 175

0 .1 0

H esidue

4 4 .7

1 .0 9 4

A p o s it i v e S c îiif f r e a c tio n was g iv e n by f r a c t io n s B, 3 and fl, 4 ; o n ly a s l i g h t r e a c t io n , a f t e r 24 hotars,\m s g iv e n by B, 6 ,

ïh s low er f r a c t io n s were in v e s t ig a t e d by h y d ro ly ­

s in g and a ttem p tin g to prepare d e r iv a t iv e s from the

73. p a r t i a l l y m ethylated re d n clo g su g a r s.

l a s s é s oa

h y d r o ly s is were heavy where th e p resen ce o f aahydro '

.t

con^ouad was aagiieated by the S o h lf f r e a c tio n and the i s o l a t i o n o f the d ia ltr o p h e n y l hydrazone o f la e v u l ic a c id i n one e s s e , a ls o agreed w ith aahydro d e r iv a t iv e s b ein g p r e s e n t i n i t i a l l y ,

f■

The h y d r o ly s is p ro d u cts vfere a ls o in v e s t ig a t e d by the p a r t i t i o n paper ctooiiatogrep hy te ch n iq u e ,

SM s

method o f a n a ly s is was d evelop ed by Oousden Gordon and M artin

f o r m ix tu res o f amino a c id s a s ob tain ed from

p r o te in s and p e p tid e s , extended to f r e e su g a rs by P a r tr id g e

[ss] and to m eth ylated red u cin g su g a rs by

H ir s t e t a l ,

[5d] .

I t in v o lv e s p la c in g a s p o t o f the

s o lu t io n to be a n a ly se d , about 8 cm ,, from the top o f a s t r i p o f f i l t e r paper { ahatraan lio. 1 ) |

th e paper i s

then hung v e r t i c a l l y from a trotxgh c o n ta in in g a m t e r ­ se tu ra ted o r g a n ic s o lv e n t , s e e d ia g . 6 , w ith the top edge o f the paper iBViiersed in th e s o l v a i t , m d p a ssin g over a su p p o rt, 3 , to p rev en t c a p illa r y sip lio n in g .

2 ie

whole i s e n c lo s e d i n a s e a le d g l a s s v e s s e l so th a t the atmosphere i s m aintained sa tu r a te d w ith w»t®r and o r g a n ic s o lv e n t v a p o u rs. A sharp l i q u i d f r o n t advances down t f e paper and the d i f f e r e n t c o n s t it u e n t s o f the sugar m ixtu re a ls o move d om a t v a ry in g sp eed s and hence become sep a ra ted in to d is c r e t e s p o ts , th e ir r e l a t i v e p o s it i o n s depending on the

74. type o f s o lv e n t la ix to re u e e â ,

fh e se p o s it io n s waj he

d e te c te d by d ryin g the f i l t e r paper a f t e r the ciiTomatogram h as run fo r a s n it a b le d iste a .c e , and then sp rayin g w ith an ap p rop riate reagen t*

in ti® p r e s e n t

m r k a lc o h o lic s i l v e r n it r a t e jso ln tio a s a s u se d , and the paper redded a t 100° f o r i5 s iin u te s , She se p a r a tio n o f the d i f f e r e n t sugar d e p iv a tiv e a depends m i n l y on th e d if f e r e n c e s in p a r t it io n c o e f f i c i e n t s , s in c e p a r t i t i o n o f each s o lu t e ta k es p la c e befeïeca *mter bound by the c e l l u l o s e end the o r g a n ic s o lv e n t moving o ver the c e ll u l o s e s u r fa c e . A dsorption p la y s some, b u t a mndi l e s s s i g n i f i c a n t , p a r t.

In g e n e r a l the most s a t is f a c t o r y s o lv e n t s

are th o se which are on ly p a r t i a l l y miscûûB wiaa tm te r , For a g iv e n s o lv e n t o iiit u r e , R, v a lu e s o f s o l u t e s , where

by c o n s titu e n t D ista n ce m v e â by s o lv e n t f r o n t

remain p r a c t ic a ll y co n sta n t and may be used to e s t a b lis h the c o n s t it u e n t s in a lulxtnro once th ey have been measured u sin ^ known oogyjounds.

H ir s t e t a l*

[56] found th a t Rp

v a lu e s o f su gars v a ried eomewhat w ith the d is ta n c e moved and they use

more co n sta n t Kg v a lu e s to c h a r a c te r iz e

su gars and m ethylated d e r iv a t iv e s D ista n ce moved by c o n s titu e n t D ista n ce moved by tetram et h y l - i- g l u c o s e A sp o t o f 10^ t e traflseth yl-B -glu cose i s run a lo n g sid e

75, the spot of solution under luvestlgation (A and B in

d la i 5* o , ) Hg values of the coustitueata of the lov^r glycoside fractious were measured using the &mm solvent mixture as these iuveotigators^

iïâ s M a a J t*

A smitple of fraction B , 1. was hydrolysed by treateeut % 1th 0,1

followed by MHOI as #ho% m ^ the hydrolysis

was followed polarimetrioally by cooling to room temperature (BO'' ) at kuowu time iatenrals and imasuriag the rotation of the soliiiicu. Increaa®d to II, ITime (hr) 0

_^ .S

I

ÜB.4 39.3141.S |45,1

H

:

1

2 .5

i i.S

2,5

3,51 5

41,5 42.5 41,5 41,6

After 5,5 hr, heating j^chliugs reaction positive (8

at 70° )

HCl vms removed by passing the solution down a column of resta "dcmoidite

Ihia realm was found to be satisfactory

for the removal of HOI used for the hydrolysis of a mixture of tri» and tetra-methyl fmthyl glucosides,

The

product was taicea up iu ether to rmmve traces of îTisterial from the resin.

Y ield , 95;.; * m e , 4B ,0^;

+46.2

Chr o ma hy a r o l ^s i s j pr o duo t j ^, Solvent 1 4C^' a-butanol, 10 Cprayj

10,

A ^l0y4.L. spot

ethanol, 49;. water, 1.,, aimmaia,

solution of fuaooaiacal silver nitrate. f a lO^ solu tiom of te trame thyl glucose was

76. used fo r refaren oo compouad and

sp o ts o f a

■^20^ aolutiloo o f product was u sed .

Ruas were

made f o r 20 h r . mt 1S°C. ‘fh ree o o a a t it u s a t s were in d icated ^ l a , l b , I c , w ith the fo llo w iu g Rg v a lu e s ; la

,

%

0 ,8 2

lb

%

0 .8 6 -0 ,8 7

Ic

Rg

1 ,0 1

•Itie Hg v a lu e o f lb v a r ie d over the wide reage 0 ,8 0 4 -0 ,8 8 b u t c o u s is t e a t v a lu e s fox l a aad l o were o b to iu e d ,

ïh© Rg v a lu e o f 1 ,0 1 su g g ested th a t

2 {3 j4 -triia et}iiy lrh a m o sô waa

2 * 5 ,4 * 6 -1©trmBiethyl-

g a la c to s e aad Sî4-diB ® tiiyirhsm aose are qw>ted [56] a s showing Eg v a lu e s o f 0 ,8 8 ,

A d d itio n a l chroRiatogrtans were

then ruii w ith a th ird sp o t o f tetraD Jsth ylgaiactoso s o lu t io n in order to compare the p o s it io n o f t h i s cos^ouad w ith l b . 2 s3 t4 :6 -te tr a r a e th y lg a l8 c to s® was f a s t e r moving th©u l b , from the r e l a t i v e l a t e a s ! t i e s and a re a s o f the s i l v e r n it r a t e s p o ts , l a s l b i l c i s roughly 2 :E ;3 .

preparation af_2i8i4-% im eth2lr% G m ose^^illda^ SXace chromatography in d ic a te d th at tr im sth y lrhamnose was p r e s e n t, 0,1268 g . was e it r a e t e d w ith petroleum e th e r a t room tem perature in ord er to sep a ra te i t to some e x t e n t from tiie two o th e r sugar d e r iv a t iv e s ; Y ield 0,0723

g,, 57^ ,

S h is product was r e flu x e d fo r

S h r. w ith 0 .0 3 1 g. dry f r e s h ly d i s t i l l e d a n ilin e and

77

3 m l. ItOE.

.

removal o f so lv e a t aad tr lta r a tlo m

w ltil petrolema eth er a c r y s t a llin e i^oduot waa ob tain ed , which formed f e a t h e r - llt o c r y s ta ls over the w a ils o f the fla s k . Y ield , 0.03& g . crude, s li g h t ly d iscolou red product; m.p. 107-109 \ iro d u ct r e c r y s t a lliz e d from a lc o t o l: eth ers petroleum e th e r ts I t l t E , f lu o w hite n e e d le s; m.p. did not depress the m.p. o f 2

110

;

the product

trlm ethylrhm m ose

•uilidejW - 130(0.3 iji CmeA C, 64.8^ ; ii.

U, S.15^ ^ offer !%Iir {«Oiler and S tr a u ss ); (m o ,3 S .6 Theory 6 4 .1 ^ 8 .B 4 , 4 .9 8 . 8 5 .1 . 3?^ractlon £ . A£.

H'°=+62 Sif.61

A sample of fraction

{ C = 2 .4 iu H o —O ^)

was hydrolysed with 0,1 D

and ^ 4 HCl as shown, the hydrolysis being followed polarimetripally• 0 .1

(h ri lo

0 .5

i'4 BC]L

(CÎ ^ 3 . 1 ,5

1

5 0 .0 5 7 .9 6 0.4

60’ ).

Ô

3

11

14

3 .3 5

56.7 55.7 51.7 5 5 .0 55.4 501) 51ja 51.2

w:57ro

After

In i; HCl

11

67

80.5

ÔC% 5 80.5

6 .5

8 .7

47 46

47

51 47

4 7 ,5

h r s , h e a tin g ,ife h lin g ’ s r e a c tio n p o s it iv e ( 1 0 ' a t

The product was n e u tr a liz e d w ith BaCOg, tuj^en down to

dryness in vacuo and eaitracted w ith e th e r . Y ield , 60^-, a fte r

0 .1

M HCl treatDient; (product on

smbsequent treatment w ith H HCl recovered in 81;.- y i e l d ) .

78.

.aie, 33.5^.

. +103

H"-+106.

£ r £ p ^ a t ^ u of_% ili4ejL 0*100 g» of tîie hydrolysed fraction was treated with 0*060 g* freshly distilled aniline and 4 ml* alcohol and boiled for 3 hrs*» after cooling* triterating with petrolenm ether* and allowing to stand for t days* e crystalline product was obtained,

me prodnct was

triturated with cold alcohol filtered and washed with a little petroleum ether M.Bt 1E6-1^7\

Yield EOmg,

{1st crop) 0,134 g, treated 0,070 g, aniline gare 50 îog, (1st crop)xa,p, 129^152

îhe anilide was re crystallized

from alcohol petroleum ether (1,1} vdiite needles 1st crop m,p, ISO ; 128" (Me, E£,3j> 2nd crop m,p. 127 ;

œ e . S3,7;â, Oirorostoiîr^j^T_of iiy d x o lis ^ d j ^ . Procedure, as for fraction B,l, ihree constituents were indicated with the following Hg values 2a

%

0,61

2b

Hg

0.82

So

Hg

0,87

A Chromatogram run with 2;S:4;6-te tramsthyIgelac toso for CHM aparison showed that this compound was faster moving than 2c,

2îSî6-trimethylglucose aad 2:Sj6-trirBethylruaaaose

are quoted [56] a s showing; Hg values of 0*81;

there is

evidence from the work of D,it,Hardy that Diannose is not

79. preseat in

Ulva poXyeacoharMej

spot t h did move at

tritueth^Iglacose In two

the sam © rate as

cliTomatogrmas, r tm for eoit^arlson, b u t m v e û slightly more slowly in a third.

F raction S. Fraction A3 became yellow after standing for abont

two d ays. A3

H"% 65

+73

{C = 3.7 in %0)

S’? V O

0 .1 sA liCl

( c = 2 .8 J I

Slin© (hr) ^ 0 .5 : M :L

:

%S%

T

1 .7 5

1

68 .3 I7 O.4 7 4 .3

2 .7 5 75 .5

5.25 7 5 .5

8 6 .8 Constant duriîig 3 h r s . h e a tin g . j

77.7 ^80.9 8 2 .8

8 3 .4

8 3 .4 1| 9 6 .8

i'e h lin g s i*eBOtioa p o s it iv e a fte r 5 h r . h ÿ â r o ly s ls , She product ^as worked up as fraction A2. Yield, ùlfp after 0 .1 HCl treatment, product oa subsequent treatment with M Eg3% recovered in 91ÿ yield.

% e, 50^.

^

(,C5

^JULj^de, fori?iatioa was attempted but only a amall anomt of very soft crystalliiie m aterial was obtained on standing, which was lost on triturating with alcohol or petrolom ether, j[solatiou of i^evullc ac^d^^2^%dinltro^he%)2l>^ra2onG. By extracting tiie Barium salts, remaining after ether extraction, with warm water and adding a solution of

1

80*

2 ;4-dialtrophemyl hydra%lae in fid* a umall m o v u t of yellow precipitate yma obtained. from OHClg* m.p. 205'’.

Thie was raorysteilllzad

Mixed m*p, with the same

derivatlTe of laevalie ncid prepared from cane sugar* 205“* of A3, was determined oryoscopically ia benzene, 0.1200 g. in 17.50 g. C,showed a depression of fz Pt of

0.161'

• M.Wt. 212.

further meth.vlatlon of l^raction A3. 1*032 g. fraction A3 was refluxed with 3 ml.

+1.0 g.

fresh dry Ag^O; 0.5 g. AggO was added after two hours* 0.5 g. AggO-»-£ ml T#1 after four hours heating* and 0.5 g, A^O after six and eight hours heating.

Ihe

product was extracted with CHClg* the extract dried wifâi tigS04 * and solvout distilled off. 1st treatment* yield 0.986 g* 53.5^ (me 3rd

"

4th 5th

* ’

0.914 g*

55.46^ ”

0.840 g*

56.4^

0.652 g*

55.9^

”

The product was di8til].6d in facuo* using a small flask with wide side arm and no fractionating coluBm; 0.523 g. main fraction was obtained, Bath T 96-101"/

0.21 em. Hg.

She pale yellow residue In the flask

was not volatile at 140'.

g l.4 4 3 2 ;

m e , S6 , 2 ;u

81. Sydrolxs^Sj, C = 1 2 .1 in 0 .1 KHCl.

0.25

lïime (3ir),

0 .5 93

1 .2 5

E.Oi 4 .0

193.5 193

Yield 58^1 {üÿdrolysis of

93

n

6 .5 '1 0 .5 j 93

93

j

tetramethyl glucoside

gave 84^ yield) 43.9^ C^. W^^g-llO (C - E.8 in M m )

-119 =^^9.0

(C = 0.8 in HgO)

Formation of__%ilide from methylated fraction A3. Ü.10 g. was refluxed for 3 hr. with 0.06 g. freshly distilled aniline in EtOH.

After removal of solvent

a small amount of crystalline product was obtained. Mpt crude product 165", after trituration with alcohol petroleum ether (1:2) Oiromato^r^hy^of j^_and_fully_ræthylated A3^ after

hYlrolysls^.^ Procedure as for fraction B.l. Three constituents v/ere indicated in fraction A3, with the following Rg values, 3a

Rg

0.47 (trace)

3b

Rg

0,62

3c

Rg

0.82

After méthylation the following Rg values were obtained.

82.

3Pa

Rg

0.89-0.91 (trace)

3Pb

Rg

1.0

SPc

Rg

1.01 (main)

*

Tiius rhamnose and glucose are present although the third spot does not correspond to any value quoted for a fully methylated sugar likely to be present, neither is it due to a trimethyl galactose (2:3:4-anilide, m.p. 168").

Fraction 4. Fraction B4 was investigated since this showed a rise of methoxyl figure, suggesting some separation of a more completely methylated biose, which was not apparent in the first fractionation, A.

iVlso

crystalline material formed during the preliminary fractionation at bath T.

144^.

On triturating a

sample of fraction B4 with dry M eO H a small amount of crystalline material was obtained, m.p.,after draining on porous tile and reorystallizing once from methyl alcohol, chloroform (1 : 1), 44^45". Fraction B4 became yellow on standing for 24 hours.

85. Sydroljiais^ In 0.2 N HCl (c = 1.3) Time, hr. : 0 ! 0.£5 -

|96.8

0,5 95

1

In NHCl 1.75

89,2 96.5

1.5

r-----" ,8,5

3.6_ 95 95,5_ 93.5

93.5

2,75

»

0,5

She product was worked up as fraction IB, using "de-acidite" B. Yield 75;^ containing a lit tle material from resin.

Clirpmato£rap^_of È^drol^sis_j)rodu£tj5_ 4a

Rg

0.54

4b

Rg

0.74

4c

H g

0.86-.87.

84,

im m riQ A T iO M

Emm

-Acidic material^ after separation as Barlnm salts was e^terified with M eO Il/H C O L (1#5^), HCl was removed aeration and

M eO H(p,69)

the ester extracted with

A oamplo of the ester fro?n methanoljFsis II was converted back to Barium salt by refluxing %ith 4< baryta, Uie solution vms bailed gently aad

^ 0*5 ml, distillate

collected which contained M eO H (oxidation to ECH Oand reBorcinol colour test),

fh e solution was made just acid

to a>ngo, concentrated somewhat at 40°, and extracted with CHCls,

A dark jÿ^llow viscous syrup was obtained 23^ O T vle,

i,e . 0.21 g. 18;&

O.U@ 4 34 ' mie -5>

Ba s a l t

E s te r

£rac tiâïiAl^d^s t i l

0.068 g. 25^ 0^ ->

f r e e a c i d +M eOH,

io n .

0 ,4 0 g . a s t e r fro m m & th a n o ly s is I I I , 5 7 .8ÿ, dtio, %ms f r a c t i o n a l l y d i s t i l l e d , ms la g a ^ m l l d i s t i l l i n g f l a s k w ith w ide s i d e arm and no f r a c t i o n a t i n g c o ltm n , m ain f r a c t i o n w as o b t a i n e d , b a t h T 146" / o , 2 0 40.2;: Qf.e,

1.4628,

M

C.1S6 g , %,

=+4 9 , There waa an

a p p r e c i a b l e r e s i d u e , a o i v o l a t i l e below 180'.

ïiie ester fraction froia methanolysis I? was obtained aimllariy and then reconverted to Barium salt and re-*estorifled in order to remove glycosidas more completely, After re-esterificatioii material was recovered in poor y i e l d ( 51 yA) b u t p a r t o f the p r o d u c t c r y s t a l l i z e d on

85, ataading for 6 days,

«

mrodgot. M pt ortide m aterial

118 \

%t af ter reo ry eta lll& etlam from a lo o h o l/p etr o le im

eth er

118-119% Botatloh.

w a t e r ,

a a c

e o iu V io ^ i

,.o

a f t e r

The product was appareatly lusoluble la

o p t i c a l

a c t i v i t y

s t a a d i n g

o v e r

c o u ld t h i s

b e

d e t e c t e d

c r y s t a l l i n e

la a a

a q u e o u s

m a t e r i a l ,

A positive iodoform reaction was given,

m e , 8 9 .8 ^ , Attempted preparation of m&lde, A solution of 0*068 g. distilled ester in 8 ml, dry l40(H was saturated with dry

and left at O '' for 8 days.

O a removal of solvent a syrup % ae obtained, . The treatcteiit was repeate

and after prolonged standing at

Z ” there was not sufficient crystalline material to isolate, but amide formation had taken place, since after repeated evaporation with M eC Mto drive off free Mg, the product yielded 11%on heating with baryta.

■■ diagram7

86*

UROmiG ACID uroiiic acid (or keto g l ^ o o u l c

acid) conteats

of the fuily methylated and chloroform inaoluble fractions of methylated polysaccharide were estimated by distillation with HCl^ according to the procedure of Dickson et al,

[57] »

îhe apparatus was modified

{Diag, 71 by the use of all glass Joints where possible, and the C% absorption inrolred oa introducing baryta in the original method was elieduated by the use of an enclosed siphoning arrangement for drawing baryta into the burette from the re sevoir, iC, by the three-way tap, J, connected to a pump, to the atmosphere and through a soda lime tube to the burette, A stream of air, freed from 0% by passage through the soda lime towers AA^ was drawn through the apparatus by a water purg> attached to the safety vessel H* containing

The sample,

0,3 g. uronic acid was placed in the reaction

flask C, with 100 ml, 13,16^7 HCl and jîorous pot;

the

height of the oil bath, B, was adjusted so that the acid level was Just below that of the o il,

fhe air

stream was passed for about twenty laiautes, and then heating of the flask commenced, approximately 20 ml, 0,2

Vthen the acid boiled,

baryta was run into the

absorbing tower G, filled with glass beads, and the rate of air flow incroascd.

The base of 0 was flanged outwards,

and the upward air stream prevented the baryta running

87. into the Bucixiier flask. F.

.

!0ie trap, D, containing

aniline, removed furfural end the trap E, contaixiing silver nitrate solution, removed any HCl not refluzed back by the condenser, or removed by i^hÆ .

£

Heating was continued for 5 hr, with the temperature of the oil bath maintained at 135-140“ and the air flow adjusted to

bubbles p e r second.

At ikm end of

this period the tower was washed down with CO g ,free water and excess baryta titrated with 0,1 lî HCl in an aÜ30sphere of nitrogen, using phaaolphthalein indicator, 5he chloroform ixisoluble material was boiled with a lit t le I p HCl to remove the sttiall amount of free carbonate present and the acid concentration then made up to 13,15^ before estimating uronic acid.

88. Hesuits. HCl 0 .0 9 7 0 m. Direct t i­ ml baryta used in tration, ml HCl = tower 10 ml barytii

ml HCl I ml HCl required sC% to neu- ! tralize,'

Blank estimation

\

£4,78

1 0 ,0

£4.70

0,08

£4,78

1 0 .0

£4.71

0,07

Blank correction =-0.08 bjI HCl

a l HCa s ccfe corrected Cor blank

Ctoeck estimation on 0.1986 g

24,78

1

ml IÎC1

s i gBl

galacturoaic acid 26,24

43.41

£1,51

108

( %eoretlo@l 106) i'ully metiiylated material (corrj for astil 0.5610 g.j 24,78

19,97

43.71

5.63

0,4314 g. 20.83

19.04

35,50

4.12

0.5005 s. 20.83

20.32

37,39

4,86

1 0 ,0

CHClglasol. material (eorr: for ash) 0.849 g.

£0,83

34.34

67,51

17,48

20,50

•'.2150 g. iisthylnted material (3p^0Me ) contains 1 g,e, C O C SI, i.e . 1 acid group per 9 (methylated auhydroheaKise) units. .. 1006 g. GHGlg insoluble material (19^ ORle) contains 1 g.e, CO0H, i.e . 1 acid group per -5 units.

89,

A im îim io h ts ïB . Autohjûrolysls of a O.Bÿ, solnÈlon of acid form polysacc­ haride, pH 3, was carried out on a boiliag water bath in B nitrogen atiaosphere,

fh© hydrolysis was followed by the

development of reducing power, using the hypoiodlt* titration Biethod of Baiter and Hulton [45} aad polarimetr1cally when the solution had cleared sufficisitly.

Both methods

indicated that a steady state was reached after 43-50 hr. heating. SCim e (hr.)

■

1

1.&5

W Slio

-

ml. 0.114 Ig -1

0,26: 14 : 20 32 & mj -72 165 -59 r47

6,5 15,3

t

40.8 43,25

m

50

-36

i---34^ 45*8 58*2 55.£

55*£

resuitiiig aolatloii \m s jellow h ro rm m û ooataiueû a light-weight brown suspeaaioa.

It was concaatrateâ to about

one third volume in vacuo at 45" in 1% ; was precipitated*

a li t t l e CaSO^

t h e solution, containing

£.1 g* organic

matter, v/as dialjsed against tlirec 500 ml* volumes of distilled water over a period of £i days.

During this time the

lïjaterial inside the dialyser darkened considerably,

lEhe

dialyse tes were concentrated at 45“ in % and gave a positive naphthoresoroinol reaction for uronic-acid, there was no marled reaction from the non-dialysable mterial, although its dark colour made results inconclusive,

-Aliquot portions of

both fractions were boiled with HCl to assess the distribution of methyl pentose and uronic acid {and/or pentose).

Of: ' -'-

*

■.?'?-• -■ ■

-

---5:

-,f

z:- -ri

.

,

z;-

i'w C w el ana cigttol.furfural EgtiiaaU 0.00418

vûmre E* = mass o f aahjdrons

rhamixose. û .02194 g . L^rhamiose h jd rate gave 0.01320 g . p p t ., la agreement w lih t h is exp reselon