Sc.

PREPARATION AND S T R U C T U R E S

O F l S U C R O S E M O N O E S T E R S

A T H E S I S

SUBMITTED TO T H E

F A C U L T Y OF PURE AND A P P L I E D

S C I E N C E S , IN P A R T I A L

F U L F I L L M E N T OF T H E R E Q U I R E M E N T S

FOR T H E D E G R E E OF

MASTER OF S C I E N C E

IN T H E

D E P A R T M E N T OF CHEMISTRY

U N I V E R S I T Y OF OTTAWA

BY

°L'o,

>* V^i X

''SHAM ^

V

J E A N - M A R C BILLY

M . S c . CANDIDATE

R . U . L E M I E U X

PROFESSOR OF CHEMISTRY

SUPERVISOR OF RESEARCH

^ BlBUOTHklUES

im URRARIES . •

S E P T E M B E R , 1 9 5 7 .

UMI Number: EC55498

INFORMATION TO USERS

The quality of this reproduction is dependent upon the quality of the copy

submitted. Broken or indistinct print, colored or poor quality illustrations

and photographs, print bleed-through, substandard margins, and improper

alignment can adversely affect reproduction.

In the unlikely event that the author did not send a complete manuscript

and there are missing pages, these will be noted. Also, if unauthorized

copyright material had to be removed, a note will indicate the deletion.

®

UMI UMI Microform EC55498

Copyright 2011 by ProQuest LLC All rights reserved. This microform edition is protected against

unauthorized copying under Title 17, United States Code.

ProQuest LLC 789 East Eisenhower Parkway

P.O. Box 1346 Ann Arbor, Ml 48106-1346

I

PREFACE

The monoes ters of sucrose p repared from the

higher fatty acids have received much at tent ion in recen t

years in view of the i r poss ible use as non-ionic d e t e r g e n t s .

The i n t e r e s t in these compounds was in i t ia ted and fos tered

by the Sugar Research Foundation, New York which has

es tabl ished grants in aid of r e s e a r c h on these compounds

in severa l consult ing and un ivers i ty l a b o r a t o r i e s . The

work presen ted in this thes i s was pa r t of this projec t and

was concerned with the study of a var ie ty of problems

which a rose in the attempt to p repa re sucrose monoes te rs

by es te r i f ica t ion of sucrose using methyl e s t e r s with po­

tass ium carbonate as ca ta lys t and N, N-dimethylf ormamide

as solvent . The main concern was with the es tabl i shment

of procedures for the isola t ion of pure reac t ion p roduc t s ,

the analysis of crude reac t ion p roduc t s , and the d e t e r m i ­

nation of the s t r u c t u r e s of the reac t ion p roduc t s . Time did

not allow a detailed study of the k inet ics and thermodyna­

mics of the r eac t i on . However, a few p re l imina ry r e s u l t s

were obtained on these m a t t e r s .

I I

I wish to express my deepest g ra t i tude to my

r e s e a r c h d i r e c t o r , Dr . R .U. Lemieux, for t ry ing to

ins t i l in me the proper approach so important in c a r ­

rying out sound scient i f ic r e s e a r c h .

I also wish to thank the Sugar Research Foun­

dation of New York for a grant in a id .

I l l

TABLE OF CONTENTS.

I . INTRODUCTION 1

1. S u c r o s e 1

2 . D e t e r g e n t s

a. Def in i t ion and C l a s s i f i c a t i o n 3

b . N o n - i o n i c D e t e r g e n t s D e r i v e d from Polyhydroxy Alcoho l s 6

c . D e t e r g e n t s D e r i v e d f rom S u c r o s e 14

3 . The S t r u c t u r a l S tud ie s on S u c r o s e E s t e r s 22

I I . EXPERIMENTAL

1. Reagen t s 29

2 . The Acy la t ion of S u c r o s e by T r a n s e s t e r i f i c a t i o n 31

3 . The Acy la t ion of S u c r o s e with M y r i s t o y l C h l o r i d e 33

4 . A n a l y t i c a l Methods

a. Sapon i f i ca t ion and F a t t y Acid Conten t D e t e r m i n a t i o n 33

b . F o r m y l Group D e t e r m i n a t i o n 35

c . Pape r C h r o m a t o g r a p h y of the S u c r o s e E s t e r s 38

d. P a r t i t i o n C h r o m a t o g r a p h y of the S u c r o s e E s t e r s on C e l i t e 42

5 . D e t e r m i n a t i o n of the Number of M y r i s t o y l Groups at the 6- and 6 ' - P o s i t i o n s in a S u c r o s e M y r i s t a t e

a. The P r e p a r a t i o n of T o s y l E s t e r s 47

IV

b . I od ina t i on of T o s y l E s t e r s 5 0 .

6 . Sodium P e r i o d a t e O x i d a t i o n s 5 1 .

7 . Reduc t ion of T o s y l E s t e r s with L i th ium Aluminum H y d r i d e 5 3 .

8 . The P r e p a r a t i o n of R a d i o a c t i v e S u c r o s e P a l m i t a t e s and the C h r o m a t o g r a p h i c S e p a r a ­t ion of a M i x t u r e of S u c r o s e A c e t a t e s 5 5 .

I I I . DISCUSSION OF EXPERIMENTAL RESULTS 5 9 .

1. The P r e p a r a t i o n s of S u c r o s e M y r i s t a t e by T r a n s e s t e r i f i c a t i o n 60-

2 . The P r e p a r a t i o n s of S u c r o s e M y r i s t a t e u s i n g the Acid C h l o r i d e 68 .

3 . The Rate of the T r a n s e s t e r i f i c a t i o n

R eac t i on 71 .

4 . The S t r u c t u r e of the S u c r o s e E s t e r s

a. The Number of M y r i s t o y l Groups at the 6- and 6 ' - P o s i t i o n s in a S u c r o s e M y r i s t a t e 7 2 .

b . The P e r i o d a t e Ox ida t ions 7 8 .

c . The Reduc t ion of the Tosy l E s t e r s 8 1 .

CLAIMS TO ORIGINAL RESEARCH 8 5 .

BIBLIOGRAPHY 8 7 .

V

LIST OF TABLES

I . S t a n d a r d i z a t i o n of the An th rone Method for the D e t e r m i n a t i o n of S u c r o s e C o n t e n t s 44

I I . C h r o m a t o g r a p h i c A n a l y s e s of the R e a c t i o n P r o d u c t s f rom the T r a n s e s t e r i f i c a t i o n of Methyl M y r i s t a t e with S u c r o s e 47

I I I . The Oxida t ion of S u c r o s e and G lucose with Sodium m e t a P e r i o d a t e 52

IV. The S e p a r a t i o n of a M i x t u r e of S u c r o s e A c e t a t e s on C e l i t e 57

V. The P r e p a r a t i o n s of S u c r o s e M y r i s t a t e by T r a n s e s t e r i f i c a t i o n 61

VI. The P r e p a r a t i o n s of S u c r o s e M y r i s t a t e s u s ing the Acid C h l o r i d e 69

V I I . The T o s y l a t i o n and Iod ina t i on E x p e r i m e n t s 73

V I I I . The Consumpt ion of P e r i o d a t e and the P r o d u c t i o n of F o r m i c Acid for the P o s s i b l e M o n o - 0 - S u b s t i t u t e d S u c r o s e s 79

IX. The P e r i o d a t e Oxida t ion of S u c r o s e M o n o m y r i s t a t e at 24 .8 C. 79

The Reduc t ions of the T o s y l a t e s of S u c r o s e and of S u c r o s e M o n o m y r i s t a t e wi th L i t h i u m Aluminum Hydr ide 82

LIST OF FIGURES

1. S t a n d a r d Curve for the F o r m y l Group D e t e r m i n a t i o n P r o c e d u r e 37

Kine t i c Run of the T r a n s e s t e r i f i c a t i o n R e a c t i o n 48

3 . The Iod ina t i on of the T o s y l a t e s of S u c r o s e and of S u c r o s e M o n o m y r i s t a t e 75

VI

ABSTRACT

S u c r o s e has been a c y l a t e d by way of m y r i s t o y l

c h l o r i d e and a l s o by t r a n s e s t e r i f i c a t i o n with m e t h y l my­

r i s t a t e u s ing p o t a s s i u m c a r b o n a t e as c a t a l y s t in N , N - d i -

methy l f o r m a m i d e s o l u t i o n . The i n i t i a l p r o d u c t s in each

c a s e were h igh ly s u b s t i t u t e d s u c r o s e m y r i s t a t e s . Under

the c o n d i t i o n s of the t r a n s e s t e r i f i c a t i o n h o w e v e r , the

p o l y e s t e r s r e a c t e d with the e x c e s s s u c r o s e p r e s e n t to

y i e ld even tua l l y s u c r o s e m o n o m y r i s t a t e s as the ma in p r o ­

d u c t . The d ime thy l f o r m a m i d e so lven t did not r e a c t ex ­

t e n s i v e l y with the s u c r o s e to form f o r m a t e e s t e r s .

T o s y l a t i o n and i o d i n a t i o n s t u d i e s have shown tha t

the l ' - t o s y l o x y group of a s u c r o s e t o s y l a t e is not r e p l a ­

c e a b l e by an iod ine a t o m , only t o sy loxy g r o u p s at the 6-

and 6 ' - p o s i t i o n s r e a c t . T h i s fact was used to show tha t

a p p r o x i m a t e l y 50 p e r c e n t of the e s t e r g r o u p s of a s u c r o s e

m o n o m y r i s t a t e p r e p a r e d by t r a n s e s t e r i f i c a t i o n a r e at the

6- and 6 ' - p o s i t i o n s of the s u c r o s e r e s i d u e . P e r i o d a t e

o x i d a t i o n s of t h e s e m o n o e s t e r s s u g g e s t tha t the o t h e r

half of the g roups occupy the 1 ' - p o s i t i o n . I n d i c a t i o n s a r e

tha t the s u c r o s e m o n o m y r i s t a t e s ob ta ined from the a c y l a ­

t ion in p y r i d i n e have a p p r o x i m a t e l y 70 p e r c e n t of the m y ­

r i s t o y l r e s i d u e s at the 6- and 6 ' - p o s i t i o n s of s u c r o s e .

-1 -

I . INTRODUCTION

Since t h i s r e s e a r c h was c o n c e r n e d with the p r e p a ­

r a t i o n of e s t e r s of s u c r o s e for p o s s i b l e u s e as d e t e r g e n t s ,

i t was b e l i e v e d d e s i r a b l e to r ev i ew b r i e f l y the v a r i e t y of

me thods which have been used to p r e p a r e e s t e r s of c a r b o ­

h y d r a t e s t o g e t h e r with the me thods of i s o l a t i o n , p u r i f i c a ­

t ion and proof of s t r u c t u r e of t h e s e c o m p o u n d s . S p e c i a l

e m p h a s i s was given to s u c r o s e and i t s d e r i v a t i v e s . The

p h y s i c a l and c h e m i c a l p r o p e r t i e s of s u c r o s e have an i m ­

p o r t a n t b e a r i n g on the p o s s i b l e a p p l i c a t i o n of t h e s e m e ­

thods for the p r e p a r a t i o n of s u c r o s e e s t e r s and the p e r ­

t i n e n t p r o p e r t i e s of s u c r o s e a r e t h e r e f o r e a l s o r e v i e w e d .

A s h o r t no te on the t h e o r y and c l a s s i f i c a t i o n of d e t e r g e n t s

i s i n c l u d e d .

1 . S u c r o s e

S u c r o s e , / t f -D-f ruc tofuranosyl o(-D -g lucopy r a n o s i -

d e , o c c u r s a l m o s t u n i v e r s a l l y t h r o u g h o u t the p l an t w o r l d .

The p r i n c i p a l s o u r c e s of c o m m e r c i a l i n t e r e s t a r e the s u ­

ga r b e e t , the s u g a r cane and the sap of m a p l e t r e e s . Su

c r o s e i s a whi te n o n - r e d u c i n g c r y s t a l l i n e d i s a c c h a r i d e

m e l t i n g be tween 160 and 188*C.(1) depend ing on the m e d i a

used for i t s p u r i f i c a t i o n . The compound has a s p e c i f i c r o -

tation,L4.jp+6 6 . 53 ( c . 26 in w a t e r ) ( 2 ) , and is h igh ly s e n -

- 2 -

s i t ive to acids and enzymes undergoing hydro lys i s in

the i r p resence to give a mixture of equal amounts of D-

glucose and D-f ruc tose . This hydro lys i s p roces s is cal led

invers ion by reason of the fact that the r e su l t ing mixture

has a negative ro t a t ion .

Levi and Purves (3) have reviewed the l i t e r a t u r e

re la t ing to the s t r uc tu r e of sucrose up to 1949, and the

numerous r epo r t s of the biochemical synthes is of suc rose

have been compiled by Hassid and Doudoroff (4) up to 1950.

The configuration of the anomeric cen te r s of suc rose were

es tabl ished by X-ray c rys t a l log raph ic analys is in 1947 (5) .

The conclusions reached in this fashion were subs tan t ia ted

by chemical means when Lemieux and Huber (6) synthesized

sucrose in 1953. Recently, Lemieux and Bar re t t e (7) have

proven the tf-D- configuration of the f ructose moiety when

they tosylated the c rys t a l l i ne 2 , 3 , 6 , 3 *, 4 ' - pen t a -O-ace ty l

sucrose (8) and detosylated the r e su l t ing t r i t o s y l p e n t a a c e -

ta te with a lka l i . The formation of 1,4;3,6 dianhydro-/^-D-

fructofuranosyl 3 , 6 - anhydro-fl(-D-galactopyranoside was

taken as chemical proof of s t r u c t u r e I for s u c r o s e . 6 fKaOH

M^Otf

- 3 -

The numbering of the carbon atoms in the suc rose molecu-

le ( I ) is that proposed by Hockett(9) in which the carbon

atoms of non-reducing d i sacchar ides containing g lucose ,

a re numbered by using plain numerals for the glucose

moiety and pr ime numerals for the non-glucose p a r t .

2 • j ) e t e rgen t s

a. Definition and Classification.— A surface a c t i ­

ve agent is one which modifies the p r o p e r t i e s of the su r ­

face layer of one phase in contact with another . In order

to effect this change in energy between the two su r f aces ,

a molecule must have two dis t inc t po r t i ons , one of a hydro­

carbon nature (hydrophobic) and the other of a polar solu

bi l izing nature (hydrophil ic) . At the in terface between two

immiscible substances the surfactant is or iented so that

the hydrophil ic port ion is toward the polar phase and the

hydrophobic portion is a t t r ac t ed by the non-polar phase .

When mult iple polar groups are p resen t in a surface a c t i ­

ve molecule , they must be located at one end of the mole­

cule . If they are a cons iderable d is tance apa r t , the de­

s i r ed or ienta t ion of the molecule at the in ter face is defea­

ted . Compounds which are exceptions to this rule a re

usually l imited as to surface act ive p rope r t i e s ( 10). It

may be seen, consequently, that the p r o p e r t i e s of a su r ­

face active agent can be var ied at will simply by changing

- 4 -

the lypophi le-hydrophi le ba lance .

The de te rgents can be divided into th ree c l a s s e s ;

the anionic_ detergents are those which have the lypophil ic

port ion as an anion, the ca^Uyaic_ de te rgents are those which

have the lypophilic port ion as a cat ion, and the non_Z±2.RL—

detergents (11) .

In the c lass of _anJ.oniLc_ de te rgents a re found the

ordinary sodium and potass ium soaps , the purely organic

soaps obtained when ni t rogenous bases a r e used to form

sa l t s with fatty ac ids , and the alkyl aryl su l fona tes . The

organic soaps are excel lent emuls i fyers and good dry

cleaning agents being soluble in organic so lven t s . Unlike

the sodium soaps which cannot be used in acid media and

in the presence of heavy metal ions , the sulfonates a re

sodium sa l t s of s trong acids and are unaffected by ions

of magnesium, calc ium, iron and other heavy m e t a l s , and

also by low pH . An example is sodium dodecylbenzenesul -

fonate.

The so called " r e v e r s e d soaps" which make up

the second c l a s s , the cat ionic d e t e r g e n t s , e l iminate all

in te rac t ions with heavy metal i ons . They pos se s s the

disadvantage however of forming p r e c i p i t a t e s with ordinary

soaps and with other long chain an ions . These de te rgen t s

5 -

find use in acid and neut ra l media . They also exhibit

marked bac te r i c ida l ac t iv i ty . The most common ones are

qua te rnary ni trogenous compounds having at l eas t one long

chain alkyl group .

The non-ionic de te rgents overcome all the p ro ­

blems encountered by the reac t ions of ionic de tergents

with undes i rable ions . There is however no case of a

s trongly hydrophil ic non-ionic functional group; that p ro ­

per ty is supplied by more than one, usually severa l mildly

hydrophil ic g roups . The main reasons for the growing

market of non-ionics is that they are excel lent wetting

agents and produce low foam, a des i rab le proper ty for au­

tomatic equipment. The non-ionics have also excel lent

cleaning p r o p e r t i e s . In this c lass of de tergents enter the

e s t e r s , e thers and th ioe thers of polyhydroxy alcohols and

polyoxyethylene condensa tes . The polyoxyethylene e s t e r s

produced from tal l oil r ep re sen t the l a rges t f ract ion of

these chemicals commercia l ly made(12). The monoglyce-

r i d e s , the fatty e s t e r s of polyglycerol , p e n t a e r y t h r i t o l ,

so rb i to l , manni tol , and of anhydro hexi tols fall into this

c lass .

It is only recent ly that fatty acid e s t e r s of suc ro ­

se have entered in the field of d e t e r g e n t s . A cons ide ra -

- 6 -

tion of the avai lab i l i ty and low cost of the raw m a t e r i a l s

enter ing in the manufacture of a sucrose fatty acid e s t e r

de te rgen t , plus the fact that such a compound would hydro-

lyze in the stomach to form normal food components has

renewed the i n t e r e s t in these compounds. Previously r e p o r ­

ted s tudies with polyoxyethylene condensates have demons-

t ra ted that for optium surfactant p r o p e r t i e s , the p re sence

of about two ethylene oxide units for every th ree carbon

atoms in the alkyl chain is needed(13). One could expect

that sucrose with eleven oxygen atoms would cont r ibute

about the same hydrophil ic effect as a polyoxyethylene

containing an equal number of oxygen a toms . An effective

surfactant derived from sucrose would then r equ i r e an

alkyl group containing about 16 or 17 carbon a toms . This

view was supported by the fact that glucose and sorb i to l

contain an insufficient number of oxygen atoms per mole ­

cule and that it was necessa ry to add oxyethylene groups

to obtain sufficient water solubi l i ty with an alkyl chain

of adequate s ize for surface act ivi ty( 14).

b . Non-ionic Detergents Derived from Polyhydro­

xy Alcohols . - Even though the synthet ic hydrophi l ic groups

have a la rge pa r t of the non-ionic f ield, the na tu ra l ly

occuring carbohydra tes have been the object of continued

r e s e a r c h in the field of e s t e r i f i c a t i o n .

- 7 -

The d i rec t e s te r i f i ca t ion of polyhydric alcohols

with fatty acids is the oldest recorded and probably the

s imples t way by which to make polyhydric alcohol e s t e r s .

It was back in 1860 that the French chemist Berthelot( 15)

t r ied to p repa re an e s t e r of sucrose by heating some suc ro ­

se with a fatty acid in a sealed tube . He obtained no r e a c ­

t ion . However, even though some es te r i f i ca t ion occurs at

ordinary t e m p e r a t u r e s , i t s p r o g r e s s is slow and long

heating or high t empera tu res are requi red to ca r ry the r e a c ­

tion to complet ion. It cannot be used therefore when the re

is danger of undes i rable chemical changes such as polyme­

r i za t ion , dehydration or c h a r r i n g . The g rea t e s t disadvan­

tage of the method is observed when polyhydric alcohols

are to be only pa r t i a l ly e s t e r i f i ed . In such c a s e s , mix­

tures of more or l e s s completely es te r i f i ed alcohols a re

formed even if a large excess of the alcohol is used . The

reason for this is that most polyhydric alcohols a re not

misc ib le with the fatty acid, even at elevated t e m p e r a t u r e s ,

while the i r e s t e r products a r e . Thus the fatty acid r e a c t s

with the es te r product in p re fe rance to the alcohol yielding

more highly es te r i f i ed products than intended.

The case of the g lycer ides has been extensively

s tudied. Bel luci( l6) reac ted one mole of g lycerol and one

© mole of fatty acid at 220 C. under a p r e s s u r e of 30 to 40

- 8 -

mm . The bulk of the fatty acid d isappeared in about two

h o u r s , but an excess of the g lycerol r ema ined . Monoesters

form in i t i a l l y , but after the f i r s t half hour d i e s t e r s p r e ­

va i led . If the reac t ion was continued after all the fatty acid

had r eac t ed , the amount of monoglycer ides again inc reased

and the unreacted glycerol was used up .

Hilditch and Rigg (17) have shown that the e s t e r i ­

fication of ten moles of glycerol with one mole of fatty

acid at 180 C. resu l ted in 70% conversion of the acid into

e s t e r after four hours . Only 46% of the e s t e r s i so la ted

were monoglycer ides . More r ecen t ly , the d i rec t e s t e r i ­

fication of methyl o(-D-glucoside was repor ted (18) . With

a one to one molar ra t io of the fatty acid to the g lucos ide , o

reac t ion t imes of ten hours at 230 C. gave d i e s t e r s as

main products .

The fatty e s t e r s obtained by t r ea t ing hexi to ls with

fatty acids at high t empe ra tu r e s are commerc ia l ly impor­

t an t . The pa r t i a l e s t e r s in p a r t i c u l a r have valuable s u r ­

face active p rope r t i e s and the i r comparat ively bland t a s t e

an freedom of toxici ty give them a wide appl icat ion in many

fields (19) .

In his a t tempts to es ter i fy mannitol with l au r i c

acid in concentra ted sulfur ic ac id , Bloor (20) obtained a

- 9 -

mannitan d i l aura te which he converted on heat ing to a

dianhydro mannitol d i l a u r a t e . With 85% phosphoric acid

as ca ta lys t , Brown (21) obtained s imi l a r anhydro d e r i v a t i ­

ves from the es te r i f i ca t ion of sorb i to l and mannitol with

fatty acids .

The above r e s u l t s show that the use of high tem­

p e r a t u r e s and acid ca ta lys t s can cause deep changes in the

alcohol mo lecu l e s . Even if improvements have been made

to bet ter the y ie lds and the quality of the products ob ta i ­

ned by the d i rec t e s te r i f i ca t ion method, the conditions of

th is reac t ion do not allow sucrose to be es te r i f i ed by this

route since sucrose is highly sens i t ive to acids and tem-

p e r a t u r e s above 100 C. will cause ca r ame l i za t i on .

The use of acid_ clUorjjdes and_a1cid J^iJ^jlj^ideis in

a large excess of anhydrous t e r t i a r y amine affords a favou­

rab le method for the e s t e r i f i ca t ion of carbohydra tes under

mild condi t ions . Usually the reac t ion is c a r r i e d out in

the cold or at room t e m p e r a t u r e .

In 1921, Hess and Messmer (22) obtained what they

cal led octapalmityl suc rose by reac t ing sucrose with an

eight molar excess of palmitoyl chlor ide in quinol ine . The

soft granular mass i so la ted was p rec ip i t a t ed from ether

by the addition of a lcohol . The melt ing point of the e s t e r

-10-

was 54.5 C. with M^+17 . 12 . They also r epor t ed a s u c r o -

se o c t a s t e a r a t e . This solid,KJ^ + 16 . 55 , was obtained as

spher i ca l m i c r o g r a n u l e s , m . p . 57 C . , from a ch loroform-

ethanol m i x t u r e .

Unsatura ted fatty acid e s t e r s of suc rose have

found applicat ion in drying o i l s , va rn i shes and a r t i f i c i a l

th reads ( 2 3 , 2 4 , 2 5 ) . Rosenthal and Lenhard (26) have p repa ­

red a polyoleate of sucrose by reac t ing suc rose with an

excess of oleyl ch lo r ide . This e s t e r , of which they do

not give any ana lys i s , was a limpid viscous oil soluble in

benzene, t e rpen t ine , and l inseed o i l .

Ha r r i s (27) found that sucrose s t e a r a t e , p r epa red

from one mole of sucrose and one mole of s t e a ry l chlor ide

in pyr id ine , was an excel lent a n t i - s p a t t e r i n g agent for m a r ­

g a r i n e . In 1934, Lorand (28) p repa red suc rose pa lmi ta te

using pa lmi t ic anhydr ide . Clayton and coworkers (29) have

recent ly introduced the use of a var ie ty of suc rose fatty

e s t e r s as addi t ives in lubr ica t ing o i l s . Although these

authors l i s t severa l fatty e s t e r s of sucrose such as a mono-

pa lmi to lea te , d i m e l i s s a t e , t e t r a o l e a t e and some mixed

e s t e r s such as a sucrose monobutyrate dioleate and a mono-

pa lmi ta te d io lea te , the p repa ra t i ve p rocedures were not

r e p o r t e d . The fatty e s t e r s of octa 2-hydroxypropyl s u c r o -

- 1 1 -

se(30) have been studied as poss ib le surface act ive agen t s .

The most widely used method for the p repa ra t ion

of polydric alcohol e s t e r s is that based upon the t r a n s e s ­

te r i f ica t ion of fatty acid e s t e r s with the polyols.By e l imi ­

nating the alcohol formed from the fatty acid e s t e r , the

r e v e r s i b l e reac t ion can be made to go to complet ion.

The most thoroughly studied example of this r e a c ­

tion is the t r an se s t e r i f i c a t i on of alkyl e s t e r s of fatty acids

with g lyce ro l . Grun and coworkers(31) repor ted conver­

sion yields of 9 4% to 96% when they reac ted ethyl s t e a r a t e o

with an excess of glycerol at 270 C. for per iods of up to

15 h o u r s . No ca ta lys t was employed. Wright and cowor-

kers(32) in 1944 found that the methyl e s t e r s of l inseed

oil fatty acids were t r a n s e s t e r i f i e d readi ly with pen tae ry -

thr i to l when lead naphthenates were employed as ca ta lys t s .

Tempera tu res of up to 280 C. were used at a tmospher ic

p r e s s u r e to obtain the convers ion .

A sys temat ic invest igat ion was conducted by Gros

and Feuge(33) in 1949 to compare the effect iveness of the

var ious known ca ta lys t s for the a lcoholysis r e a c t i o n . Of

the numerous ca ta lys t s tudied, they found that barium hy­

droxide octahydrate , l i thium hydroxide monohydrate , sodium

ethoxide and sodium hydroxide were the most e f fec t ive .

-12-

The reac t ion of 0.33 moles of g lycerol and one mole of o

methyl fatty e s t e r s at 180 C. for two hours with six mi l -

l imoles of sodium ethoxide gave the following product :

6.7% monoglycer ides , 28.5% dig lycer ides , 35.5% t r i g l y ­

c e r i d e s , 28.6% methyl e s t e r and 0.68% free fatty ac id .

All the r eac t ions seemed to come to equi l ibr ium after 70%

convers ion . By employing an excess of methyl fatty e s ­

t e r s , 90% convers ions to t r i g l y c e r i d e s were obta ined .

The ethyl fatty e s t e r s were found to r eac t l e ss readi ly

than the methyl e s t e r s .

Only a few at tempts to rep lace the g lycerol of

t r i g lyce r ide s by higher boiling polyhydric alcohols have

been r epo r t ed . In the p resence of sodium methoxide, o l i ­

ve oil and t r i s t e a r i n have been t r a n s e s t e r i f i e d with man-o

nitol by ra i s ing the t empera tu re to 275 C. under a p r e s ­

sure of 14 mm. to remove the g lycerol by d i s t i l l a t ion

(34 ,35) . Burre l l (36) has studied the t r a n s e s t e r i f i c a t i o n

of a t r i g lyce r ide (soybean oil) and p e n t a e r y t h r i t o l . The

naphthenates of bar ium, cadmium, ce r ium, ca lc ium, l ead ,

l i th ium, s t ront ium and zinc were effective c a t a l y s t s .

Attempts to p repa re the monooleate of methyl 0(-D-

glucoside by heating the glucoside with olive oil at 225 C.

with sodium methoxide ca ta lys t finally gave an anhydro de-

- 1 3 -

r iva t ive of the e s t e r expected (37) . More r e c e n t l y , the

same problem was solved by t r a n s e s t e r i f i c a t i o n with methyl

oleate (38) . A 72% yield of methyl 0(-D-glucoside monoolea-

te was obtained after r eac t ing 0.2 moles of the ca rbohydra -o

te with 0.1 mole of methyl oleate at 230 C. for 30 minu te s .

A modification of the t r a n s e s t e r i f i c a t i o n reac t ion

that has rece ived l i t t l e a t tent ion is the e s t e r - e s t e r in te r -

change r eac t i on . Konen, Cox, and Clocker (39) obtained

pure t r i e l e o s t e a r i n by reac t ing th ree moles of neu t ra l me­

thyl e l eos t ea r a t e with one mole of anhydrous neu t ra l t r i a -

cetin in which 0.05 percent of dry sodium methoxide was

d i sso lved . The reac t ion was conducted at 60 to 100 C.

under vacuum .

Of the few neut ra l and acid ca t a lys t s (sulfonic

acids and hydrogen chlor ide) s tudied, it is s ta ted that they

a re too weak or inef fec t ive . On the other hand, the a lka­

line ca ta lys t s have proven excellent for the t r a n s e s t e r i f i ­

cation r e a c t i o n . In gene ra l , all a lkal ine substances capa­

ble of forming soaps as well as the soaps themselves are

the s t ronges t and most frequently used t r a n s e s t e r i f i c a t i o n

c a t a l y s t s . The most widly used are the a lkal i a lkoxides ,

a lkal i hydroxides and the alkal i carbonates (40) .

-14-

c. Detergents Derived from Sucrose . Although

the p repa ra t ion of fatty acid e s t e r s of sucrose by the acid

chlor ide method has been the p r e f e r r ed procedure during

the past 40 y e a r s , the t r a n s e s t e r i f i c a t i o n of suc rose with

methyl fatty es te r has recent ly been receiving cons idera

tion as a commercia l approach to the p repara t ion of these

e s t e r s ( 4 1 , 42) . The main reason for this choice is one of

cos t , while the acid chlor ides are expensive, the methyl

fatty e s t e r s are easi ly obtained from natura l fats and oils .

However, the re a re numerous d i f f i cu l t i es . F i r s t , s u c r o ­

se cannot be heated for prolonged per iods at t empe ra tu r e s

o exceeding 100 C. without ca rame l i z ing . At safe operat ing

t empera tu re s the solubi l i ty of sucrose in fat is neg l ig ib le .

It is therefore neces sa ry to employ a mutual solvent which

does not decompose or enter into the r eac t ion .

In 1956, F . D . Snell and coworker s (4 l ) published

a p repara t ion of sucrose fatty e s t e r s by the t r a n s e s t e r i f i ­

cation reac t ion in which they employed N,N-dimethyl for­

mamide (hereaf te r abrevia ted DMF) as a mutual solvent

for the sucrose and the methyl fatty e s t e r . Their f i r s t o

p rocedure consis ted in heat ing, for th ree hours at 60 C . ,

t h ree moles of sucrose and one mole of methyl s t e a r a t e

dissolved in four l i t e r s of DMF. A 0.2 molar level of so­

dium methoxide ca ta lys t was used . Under these condi t ions ,

15-

25 percent of the methyl s t e a r a t e was converted to s u c r o ­

se e s t e r . Approximately the same yield was obtained when

a g lycer ide e s t e r replaced the methyl e s t e r . It is c lea r

that the above conditions did not take advantage of the fact

that the methanol l ibe ra ted during the t r a n s e s t e r i f i c a t i o n

is vola t i le and that removing it would cause the equi l ibr ium

reac t ion to go to complet ion.

The second procedure descr ibed by Snel l (4l) and

the p re f e r r ed one, involved the use of th ree moles of su­

c rose to one mole of a methyl fatty e s t e r , with about 0.1

mole of an alkal ine ca ta lys t and sufficient DMF to d i s so l ­

ve the reac tan t s complete ly . Potassium carbonate was

found to be a sui table catalyst . . Dimethyl sulfoxide was

also used as solvent , but DMF was found s u p e r i o r . Potas ­

sium carbonate is more soluble in DMF than a re calcium

and magnesium hydroxides . Sodium carbonate is the l ea s t

soluble of the four. All of these ca ta lys t s a re insoluble

in dimethyl sulfoxide(43). With sodium methoxide as ca ta ­

lys t , the use of t empera tu re s sufficiently high for rapid

s t r ipping of the vola t i le alcohol resu l ted in undes i rab le

side r e a c t i o n s .

The p repara t ion of sucrose s t e a r a t e is descr ibed

as follows. Employing completely dry m a t e r i a l s , the su-

-16-

c rose was dissolved in the DMF (3 .3 ml . of DMF per g. of

sucrose) by heat ing with vigorous ag i ta t ion . The methyl

s t e a r a t e and the potassium carbonate ca ta lys t were then o o

added and the reac t ion mixture kept at 90 to 95 C. at 80

to 100 mm. of p r e s s u r e . A six plate f ract ionat ing column

was sui table for s t r ipping the methanol from the sys t em.

After nine to twelve h o u r s , pa r t of the DMF was d i s t i l l ed

and the res idue was dried under vacuum. Analysis of

the react ion mixture after th ree hours of t r a n s e s t e r i f i c a ­

tion showed that the monoes te rs and d i e s t e r s were p resen t

in approximately equimolar p r o p o r t i o n s . On fur ther hea t ­

ing, the sucrose reac ted with the d i e s t e r s to form mono­

e s t e r s since after six hou r s , the molar r a t io of monoes te r s

to po lyes te r s was about 2 to 1, and 23.5 to 1 after twelve

h o u r s . It appears therefore that the monoes te rs in i t i a l ly

formed were es te r i f i ed more rapidly than the sucrose p r e ­

sen t . However, the conversion of d i e s t e r s to monoes te rs

resu l ted in a product containing mainly m o n o e s t e r s . The

amount of soap increased gradua l ly . After twelve hou r s ,

almost 20 percent of the potass ium carbonate p resen t had

been converted to soap . All the methyl s t e a r a t e had

reac ted after four to six hours .

Using a lower level of DMF had no effect on the

rapid in i t ia l e s te r i f i ca t ion but slowed down the subsequent

-17-

convers ion of d i e s t e r to monoes te r . For example, the

molar ra t io of monoester to d i e s t e r was repor ted as being

3 . 1 to 1 after seven hours but only 4.5 to 1 after a total

of fourteen hours when 2.3 ml . of DMF were used per g.

of sucrose in the r eac t ion . The major effect of inc reas ing

the concen t ra t ion of the ca ta lys t was the formation of

more soap as seen by the fact that when the concentra t ion

of potassium carbonate was increased sevenfold, approxi ­

mately 50 percent of the methyl s t e a r a t e or ig ina l ly p resen t

was converted to potassium s t e a r a t e after twelve h o u r s .

In a recent publ ica t ion , Snell and Osipow(42)

s ta ted that after the methyl es te r has been converted to

sucrose e s t e r s by a procedure ident ical to the one des ­

cribed above, addition of some water to the react ion mix­

ture will speed up the conversion of po lyes t e r s to mono­

e s t e r s . Thus, after a reac t ion las t ing from th ree to six

hours , maintaining the level of water in the mixture o

between 0.1 to 0.5 percent at 90 C. for an addit ional two

hours resu l ted in the rapid conversion of d i e s t e r s to mono­

e s t e r s .

The data given above by Snell and coworkers w e «

made poss ib le by an analyt ica l p rocedure developed by

these workers to study the mixtures obtained from the

-18-

p repa ra t ion of sucrose e s t e r s ( 4 1 ) . Their scheme of ana­

lys is was as fol lows.

Upon completion of a reac t ion run, a sample of o

the mixture was dried in a vacuum at 100 C. until cons­

tant in weight . Part of this sample was par t i t ioned bet ­

ween n-butanol and 10% aqueous sodium ch lo r ide . Unreac-

ted sugar is in the water l a y e r . The optical ro ta t ions of

both phases were de te rmined . An aliquot of the butanol

ayer was t i t r a t ed to determine soaps . Another aliquot

of the butanol layer was taken to dryness to de termine so­

l i d s . Unreacted methyl e s t e r was determined by saponi­

fying the dry product , d i s t i l l ing the methanol and de te r ­

mining it co lo r ime t r i ca l ly with chromotropic acid(44) .

From the rotat ion of the water phase , the percentage su­

c rose p resen t in the or ig inal mixture was ca lcu la ted .

From the observed rota t ion of the butanol layer was ca l ­

culated the re la t ive amounts of mono and d i e s t e r s . This

l a s t operat ion r equ i r e s the knowledge of the specific r o ­

ta t ions of pure sucrose monoes ters and d i e s t e r s . The

values of the l a t t e r were repor ted as being less accura te

than those of the fo rmer . This analyt ical p rocedure was

designed as a rout ine tool to assay reac t ion products r a ­

p id ly . It does not compr ise the isola t ion and analysis of

pure p roduc t s .

-19-

Several methods of pur i f ica t ion for the sucrose

monofatty e s t e r s were outlined by Snell and coworkers

( 4 1 , 42). Of these , two are designed for the i so la t ion of

crude e s t e r s for commercia l use while the th i rd procedu­

re leads to pure m o n o e s t e r s .

Procedure 1. After the reac t ion , the solvent was

removed and the dry r e s i d u e , which contained about 54 p e r ­

cent sugar , 1 to 2 percent potassium carbona te , and about

45 percent sucrose e s t e r , was dissolved in three to four

t imes i ts weight of water , and 5 percent sodium chlor ide

(based on the water) was added. The mixture was then hea-o

ted to 90 C. and maintained at this t empera tu re until the

sugar e s t e r had completely l aye red . The curd was then

withdrawn and d r i ed . It contained 80 to 85 percent by

weight sucrose e s t e r with the remainder mainly sugar and

s a l t .

Procedure 2 . - The reac t ion m a s s , from which

most of the DMF had been removed, was heated with ethy

lene d ichlor ide and f i l t e red while hot to remove the s u c r o ­

se , the ca t a lys t , and smal l amounts of soap . The f i l t r a t e

was then cooled to room t empera tu re and f i l t e r ed . The

recovered f i l t r a t e contained any unreac ted methyl e s t e r

and d i e s t e r s . The washed f i l t e r cake was e s sen t i a l ly pure

-20-

suc rose m o n o e s t e r s .

Procedure 3 . - The complete removal of sugar and

sa l t was accomplished by par t i t ion ing the sol id , obtained

after d i s t i l l a t ion of the DMF , between n-butanol and aque­

ous sa l t so lu t ion . Dis t i l l a t ion of the butanol r e su l t ed in

a product containing about 90 percent sucrose monofatty

e s t e r . The remainder was soap and sucrose p o l y e s t e r s .

This product was r e c r y s t a l l i z e d from acetone to give pure

sucrose m o n o e s t e r s .

The evaluation of the surface act ive p rope r t i e s

of the sucrose monoes ters derived from l a u r i c , m y r i s t i c ,

pa lmi t i c , s t e a r i c and oleic acids has shown that these com­

pounds a re emulsifying agents and good de tergents (45) .

Surface and in te r fac ia l tension measurements done between

concent ra t ions of 1.0 to 0.05 percent show that all the

above e s t e r s lower the surface tension against Nujol, to

between 8.4 and 5.0 dynes per cm. This is s l ight ly be t te r

in lowering the surface tensi-on than ta l l oil polyoxyethy­

lene condensate and sl ight ly l ess effective than sodium do-

decylbenzenesulfonate . Wetting p r o p e r t i e s studied by the

Draves (46) t e s t indicated that the sucrose e s t e r s were only

fair wetting agen t s . Sucrose l au ra t e was somewhat supe­

r i o r to the other e s t e r s according to this method of evalua-

- 2 1 -

t ion .

The sucrose e s t e r s were buil t for heavy duty de-

te rgency by adding 80 percent by weight of bas ic and neu­

t r a l sal t b u i l d e r s , and were evaluated separa te ly for soi l

removal and for soil redeposi t ion . The r e s u l t s indicated

that the de te rgents from sucrose were equivalent to ta l l

oil polyoxyethylene condensate and sodium dodecylbenzene-

sulfonate in soft wa te r , but that the ta l l oil polyoxyethy­

lene condensate was infer ior to the sucrose e s t e r s and

sodium dodecylbenzenesulfonate in hard w a t e r .

The foaming p rope r t i e s of the built sugar e s t e r s

were measured by the method of Ross and Miles (47) . The

p rocedure involves measurements of foam heights produ­

ced under s tandard condi t ions . Resul ts indicated that su­

c rose l aura te and myr i s t a t e are moderate to low foamers ,

while sucrose pa lmi ta te and s t e a r a t e a re low foaming

agen t s . A study of the emulsions p repared with s i l icone

oil and minera l oil and aqueous solut ions of suc rose pa l ­

mi ta te containing varying amounts of g lycerol monos tea ra -

te indicated that sucrose pa lmi ta te is sufficiently hydrophi­

l ic to r equ i re the p resence of a lypophil ic agent for good

emuls i f icat ion of non-polar o i l s .

The s tab i l i ty of the sucrose e s t e r s was d e t e r m i -

- 2 2

ned by aging aqueous solutions of these compounds con­

taining sodium t r ipolyphosphate - A 0.5 percent solution

of sucrose s t e a r a t e having a pH of 9.5 showed 8.9 and

14.5 percent hydrolys is after aging for one and four hours o

at 60 C . , r e s p e c t i v e l y . The s tab i l i ty in acid solution was

studied by boiling a 0.1 percent solution of sucrose s t e a r a ­

te in 0 .1 N HC1. After th i r ty minutes , 6.9 percent of

the e s t e r had hydrolyzed. Feeding studies on r a t s showed

that diets containing 10 percent sucrose monos teara te cau­

sed no de le te r ious symptoms after one month.

3 • The_Stru_ctural Studies on Sucrose Es t e r s

Unlike the fully subst i tu ted sucrose der iva t ives

whose s t r u c t u r e s neces sa r i l y follow from that of s u c r o s e ,

the re are a l a rge number of poss ib le posi t ional i somers

for a sucrose der iva t ive with any given degree of subs t i -

t ion . The problem of determining the percentage subs t i ­

tution of a group on each of the posi t ions of sucrose for

a monoderivat ive is a complex one. The only detai led

s t r uc tu r a l study of a sucrose monoester repor ted in the

l i t e r a t u r e is that of sucrose 2-phosphate prepared by

t r ea t ing sucrose with phosphorous oxychloride in lime

water (48) . The sucrose monophosphate was hydrolyzed in

oxalic acid solution to give glucose phosphate , f ructose

and small quant i t ies of f ructose phosphate and free gluco-

- 2 3 -

s e . Per iodate oxidation showed that the phosphate group

was at tached to the second carbon of g lucose . The s t r u c ­

tu re of sucrose phosphate was then considered to be suc ro ­

se 2-phosphate mixed with a small quantity of another phos­

phate with the phosphate group in the f ructose moiety .

The separa t ion of the components of a mixture of

a monosubst i tuted sucrose der iva t ive into the posi t ional

i somers is a difficult t a sk . A s imi la r problem was pa r ­

t ia l ly solved by Ass el ineau(49), who studied the pa r t i a l

e s t e r i f i ca t ion of methyl 0(-D - glucoside and glucose with

palmitoyl chlor ide in py r id ine . Reacting one mole of the

acid chlor ide with one mole of the s u g a r s , the products

i so la ted were separa ted on alumina and shown to contain

only 55 and 45 percent of the 6 palmitoyl der iva t ives of

glucose and of methyl c(-D-glucoside, r e spec t ive ly . The

remainder was separa ted into di , t r i , and t e t r apa lmi ta tes .

The s t r u c t u r e of some of these products was proven by pe­

r ioda te and lead t e t r a a c e t a t e oxidat ions , methylation s tu­

d i e s , and also by the synthesis of some of the i somers

using pa r t i a l l y blocked glucose and methyl 0(-D-glucoside ,

for comparison p u r p o s e s .

The separa t ion of a mixture of the pa r t i a l l y subs­

t i tu ted der iva t ives of sucrose promised to be a more dif-

- 2 4 -

ficult task in view of the poss ib i l i ty of a g r e a t e r number

of components being p r e s e n t . It is therefore unders tan­

dable that the only a t tempts to determine the s t r u c t u r e s

of the posi t ional i somers in a sucrose monoderivat ive

were c a r r i e d out on the m i x t u r e s . The p rope r t i e s of the

sulfonate e s t e r s promised useful in this r e s p e c t .

The p - toluenesulf onyl ( tosyl) and the methanesul -

fonyl (mesyl) e s t e r s exhibit ce r ta in unique c h a r a c t e r i s t i c s

which make them of grea t importance in synthet ic and ana­

ly t ica l carbohydrate chemi s t ry . Prepara t ion of the sulfo­

nates i s accomplished by t rea tment of a carbohydrate with

a pyr idine solution of an aryl or alkylsulfonyl chlor ide

(R-SO^-Cl) or under Schotten Bauman condit ions(50) . Un­

der these conditions all the hydroxyl groups may be e s t e ­

r i f ied except those on the reducing carbons which a re r e ­

placed by hal ide a toms . Thus glucose gives t e t r atosylglu -

cosyl ch lo r ide . The p r imary hydroxyl groups are more

eas i ly es te r i f i ed than the secondary hydroxyls(5 1, 52 , 53) .

The Schotten Bauman reac t ion conditions for the p r e p a r a ­

tion of the benzenesulfonates of sucrose were studied by

Menalda(54) in 1930 .

Only a few r epo r t s concerning the t r ea tmen t of

sucrose with p- toluenesulf onyl chlor ide a re found in the

2 5 -

c h e m i c a l l i t e r a t u r e . One of t h e s e was the t r e a t m e n t of 0 .01

mole of s u c r o s e with 0 .03 m o l e s of t o s y l c h l o r i d e in p y r i ­

d ine for t w e n t y - f o u r h o u r s to y i e ld an amorphous t r i - O - t o -

sy l s u c r o s e ( m . p . 66-69 C . ) having a s p e c i f i c r o t a t i o n |_o J

+ 42 .35° ( c . 2 . 4 in CHCl j ) (55 ) . Th i s p r o d u c t was undoub­

t ed ly a complex m i x t u r e but Hocke t t and Zief p o s t u l a t e d

t h a t due to the g r e a t e r e a s e of t o s y l a t i o n of p r i m a r y h y d r o ­

xyl g r o u p s , the e s t e r was ma in ly a 1 *, 6 , 6 * - t r i -O - t o s y l

s u c r o s e .

The to sy loxy g roups which e s t e r i f y p r i m a r y h y d r o -

xy l s may be r e p l a c e d by an iod ine atom when the e s t e r is

h e a t e d in an ace tone so lu t i on of sodium i o d i n e . The d i f fe ­

r e n c e in e a s e of r e p l a c e m e n t of t o sy loxy g roups e s t e r i f i e d

with p r i m a r y and s e c o n d a r y a l c o h o l i c g r o u p s i s used to m e a ­

s u r e q u a n t i t a t i v e l y the p r i m a r y g roups in a compound(56 , 57) .

In 1944, Raymond and S c h r o e d e r ( 5 8 ) r e a c t e d 175

mM of s u c r o s e d i s s o l v e d in p y r i d i n e with 525 mM of t o s y l

c h l o r i d e . The r e s u l t i n g t r i - O - t o s y l s u c r o s e was t r e a t e d

with 58 g . of sodium iod ide (386 mM) in a c e t o n e s o l u t i o n .

The p r o d u c t of t h i s r e a c t i o n ana lyzed as a d ideoxyd i iodo

m o n o - O - t o s y l s u c r o s e . T h e s e a u t h o r s a s s u m e d tha t the

t h r e e p r i m a r y a l c o h o l i c g roups of s u c r o s e w e r e e s t e r i f i e d

in the o r i g i n a l s u c r o s e t r i t o s y l a t e . A c o n s i d e r a t i o n of

-26-

the hindered na ture of the l ' - t o sy loxy group of the suc rose

e s t e r led these workers to bel ieve that the l ' - t o sy loxy

group would probably r e s i s t replacement by an iodine atom.

For this r ea son , they postula ted that the iodo der iva t ive

was 6, 6 ' -d ideoxydi iodo-1 ' -O- tosy l s u c r o s e . It is impor­

tant to note that the conditions employed by these workers

do not prove the r e s i s t a n c e of the l ' - t o sy loxy group to r e ­

p lacement , since only 2.2 moles of sodium iodide were used

per mole of sucrose t r i t o s y l a t e during the reac t ion in ace ­

tone .

Tosyloxy groups at secondary carbon atoms usua l ­

ly remain unaffected by the sodium iodide in acetone t r e a t ­

ment unless they are contiguous to a s imi la r group e s t e r i ­

fied with a p r imary hydroxyl (59) . When the l a t t e r condi­

tion e x i s t s , both groups may be removed with the forma­

tion of a double bond. Creat ion of a double bond also may

occur when there is a free hydroxyl adjacent to a tosyloxy

group at a p r imary posi t ion as in 6- tosylglucofuranosides

(60) . From the l i t e r a t u r e a l ready desc r ibed , it can be

said with ce r t a in ty tha t , since there are no secondary to ­

syloxy groups adjacent to p r imary ones in sucrose tosy la ­

t e s , the poss ib i l i ty of double bond formation during the

t r ea tmen t of these e s t e r s with sodium iodide in acetone

is e l imina ted .

2 7 -

F o r t h e i r s t r u c t u r a l s t u d i e s on s u c r o s e m o n o l a u -

r a t e ob ta ined by t r a n s e s t e r i f i c a t i o n , Snel l and c o w o r k e r s

(61) p r e p a r e d a t o sy l e s t e r of the m o n o l a u r a t e c o n t a i n i n g

3 . 1 5 t o s y l g r o u p s p e r m o l e c u l e of l a u r a t e e s t e r . T r e a t ­

men t of t h i s t o sy l d e r i v a t i v e with a 2 . 6 4 m o l e s of sodium

o i od ide in a c e t o n e at 105 C. for 2 . 5 h o u r s gave a sodium

t o s y l a t e r e c o v e r y i n d i c a t i n g tha t 1.43 t o sy loxy g roups

p e r mo le had been r e p l a c e d by iod ine a t o m s . S i m i l a r

t r e a t m e n t with four m o l e s of sodium iod ide for 16 h o u r s

r e s u l t e d in 1.63 t o s y l g roups be ing r e p l a c e d . A s s u m i n g

t h r e e r e p l a c e a b l e to sy loxy g r o u p s to be p r e s e n t on s u c r o s e

t o s y l a t e , t h e s e w o r k e r s conc luded tha t s i n c e low r e c o v e ­

r i e s a r e not uncommon , the above i o d i n a t i o n r e s u l t s sug ­

g e s t e d tha t the l a u r y l r e s i d u e s occup ied only p r i m a r y hy­

d r o x y l g r o u p s in the s u c r o s e m o n o l a u r a t e s t u d i e d . T h u s ,

t hey d i s r e g a r d e d the fac t po in ted out by Raymond and

S c h r o e d e r (58) t h a t the l ' - t o s y l o x y group of a s u c r o s e t o ­

s y l a t e is h i n d e r e d and p r o b a b l y unchanged by the sodium

iod ide t r e a t m e n t .

The i n v e r s i o n of s u c r o s e m o n o l a u r a t e in 0 .5 N

o x a l i c ac id led Snel l and c o w o r k e r s (61) to conc lude tha t

75 p e r c e n t of the l a u r a t e g roup was on the g l u c o s e mo ie ty

of the s u c r o s e m o n o l a u r a t e . T h i s s t a t e m e n t was b a s e d

s o l e l y on the v i s u a l e x a m i n a t i o n of p a p e r c h r o m a t o g r a m s

- 2 8 -

done on the inver ted p r o d u c t s . The per ioda te oxidation of

the same suc rose monolaurate r e su l t ed in an uptake of

2.915 moles of oxidant with the l ibera t ion of 0.682 moles

of formic ac id . Since subst i tu t ion at the p r imary pos i ­

t ions only can give a sucrose der iva t ive consuming th ree

moles of pe r ioda te , the oxidation of the monolaurate p ro ­

vided evidence to the effect that the laury l groups were

indeed at tached almost exclusively at p r imary pos i t i ons .

Some uncer ta in ty was introduced however by the low r e c o ­

very of formic acid ( theore t i ca l value is one mole) .

In summary, Snell and coworkers(61) concluded

that the sucrose monolaurate p repared by t r a n s e s t e r i f i c a ­

tion was subst i tu ted almost exclusively at the p r imary

pos i t i ons , and that 75 percent of the l aura te group was on

the 6 posi t ion of the glucose moiety .

-29-

I I . EXPERIMENTAL

1. R eagents

The N,N-dimethyl formamide (DMF) employed

throughout th is work was purchased from Eastman Organic

Chemicals ( b . p . 152 154 C . ) . Azeotropic removal of any

water p resen t was effected by adding between one-quar t e r

to one volume of dry benzene per volume of DMF followed

by d i s t i l l a t ion at a tmospher ic p r e s s u r e . The fract ion col-

0 o

lected between 152 and 154 C. had a r e f rac t ive index of

1.4292. The recorded values for DMF a re ; boiling point: o

153 C. and re f rac t ive index: 1.42938. This solvent could

be d i s t i l l ed from phosphorous pentoxide but no advantage

was gained by doing s o .

The methyl myr i s t a t e employed throughout th is

work was p repared from myr i s t i c acid (Eastman Chemicals 0 o m . p . 52 -53 C.) by es te r i f i ca t ion with an excess of dry

methanol using concentra ted sulfuric acid as c a t a ly s t .

After refluxing for one hour , the e s t e r layer was s epa ra ­

ted from the excess methanol by adding water and diethyl

e t h e r . The ether phase was dried with anhydrous sodium

sulfate before d i s t i l l a t i o n . The d is t i l l ed methyl m y r i s t a -

te had the following p r o p e r t i e s ; boiling point: 147 C. at

6 m m . , r e f r ac t ive index: 1.4371, saponif icat ion equiva-

- 3 0 -

o o lent : 240 g. and melt ing point: 18 -19 C. The recorded

o constants for methyl myr i s t a t e a r e ; boil ing point: 155-7 C.

o

at 7 m m . , melt ing point: 18.5 C . , and molecular weight:

242.4 g.

The myr is toyl chlor ide was prepared from m y r i s ­

t ic acid and thionyl chlor ide in benzene solution according

to the p rocedure of S .T . Bauer (62) . The m y r i s t i c acid

(0 .3 moles) was dissolved in 75 ml . of dry benzene by s t i r -o

r ing at 50 C. in a 500 ml . th ree necked f lask . The thionyl

ch lor ide (0 .33 moles) was then added dropwise into the s t i r -o o

red so lu t ion . The mixture was heated at 55 to 60 C. for

two h o u r s . Upon removal of the solvent , much c r y s t a l l i ­

zat ion took place indicat ing that some acid was p r e s e n t .

Another 0.33 moles of thionyl chlor ide were added and the

mixture was refluxed for 3/4 h o u r s . The benzene and the

excess thionyl chlor ide were removed under a vacuum and

the myr i s toy l chlor ide was d i s t i l l e d . An 85 percent yield o *

of pure compound was col lected between 155 and 156 C. at o

8 mm. of p r e s s u r e , which had a melt ing point of -1 C. The recorded constants for myr is toyl chlor ide a r e ; boiling

# © o

point : 155 -157 C. at 7 mm. and melt ing point: -1 C.

The pyr idine employed for the tosyla t ions and acy-

la t ions was the anhydrous reagent grade solvent having a

- 3 1 -

o

b o i l i n g po in t r a n g e of 1.4 C. It was d i s t i l l e d from p h o s ­

p h o r o u s p e n t o x i d e . The p - t o l u e n e s u l f o n y l c h l o r i d e , m . p .

o 0 66 -68 C . , was the h i g h e s t p u r i t y compound o b t a i n a b l e .

The d i e t h y l e t h e r and the t e t r a h y d r ofur an employed for the

l i t h i u m a luminum h y d r i d e r e d u c t i o n s w e r e d r i e d over s o ­

dium m e t a l and d i s t i l l e d f rom l i t h i u m a luminum h y d r i d e

i m m e d i a t e l y b e f o r e u s e .

Us ing c o m p l e t e l y d ry ground g l a s s equ ipment and

r e a g e n t s , 179 .5 g . of s u c r o s e (525 mM) w e r e d i s s o l v e d in

600 m l . of d ry DMF in a l i t e r round bot tom f lask by

h e a t i n g and s h a k i n g the m i x t u r e - The me thy l m y r i s t a t e

(175 mM) and the anhydrous p o t a s s i u m c a r b o n a t e ( 1 7 . 5

mM) w e r e added to the s u c r o s e s o l u t i o n . The r e a c t i o n

f l a sk was then f i t t ed with a V i g r e u x f r a c t i o n a t i n g c o l u m n .

At the top of t h i s co lumn , an elbow was i n s e r t e d which was

in t u r n c o n n e c t e d to a vacuum a d a p t e r f i t t ed with a 200 m l .

round bo t tom f l a s k . A s o u r c e of c o n t r o l l e d vacuum was

c o n n e c t e d at t h i s p l a c e .

F o r the t r a n s e s t e r i f i c a t i o n , the r e a c t i o n f l a sk

was i m m e r s e d in a wax bath kept c o n s t a n t at 90 C . I 1 C.

A p r e s s u r e of be tween 55 and 65 m m . of m e r c u r y was n e ­

c e s s a r y to keep the r e a c t i o n m i x t u r e r e f l u x i n g s t r o n g l y .

- 3 2 -

A Car tes ian manostat was used to maintain the reac t ion

p r e s s u r e constant between i O . 5 mm. of the adjusted va lue .

Under those condi t ions , the ca ta lys t was usually comple­

tely dissolved after six h o u r s . The t empera tu re of the e o

refluxing mixture was constant between 77 -79 C.

After completion of the r eac t ion , the f rac t iona­

ting column was replaced by a Claisen head and the solvent

was removed in vacuo. For this d i s t i l l a t i on , the reac t ion o

flask was kept immersed in a bath maintained at 90 C. The

vacuum was gradual ly inc reased until the res idue could be

dr ied at full oil pump vacuum. The reac t ion cake was then

dissolved in a one to one mixture of n-butanol and wate r ,

using approximately four ml . of this mixture of solvents

per g. of reac t ion product . Sodium chlor ide was added to

form a 5 percent aqueous solution and the n-butanol layer

which separa ted rapidly was washed th ree to four t imes

with half volumes of 5 percent aqueous sal t so lu t ion . Each

of these aqueous phases were in turn equi l ibra ted with a

second volume of n -bu tano l . The combined organic pha­

ses were then c la r i f ied with anhydrous sodium sul fa te , f i l ­

t e r e d , and the crude e s t e r product obtained by removing o

the solvent in a vacuum at 60 C. Ident ical r e s u l t s were

obtained when methyl ethyl ketone was used in the place

of n-butanol in the above i so la t ion p r o c e d u r e .

- 3 3 -

3 • T J l ^ ^ c y J ^ j j ) n _ o j ^ S u c r o s e w i t ^ M y r i s t o y l C h l o r i d e .

To the 5 p e r c e n t s u c r o s e s o l u t i o n in p y r i d i n e kept

at i c e ba th t e m p e r a t u r e , was added s lowly with s t i r r i n g

the m e a s u r e d amount of m y r i s t o y l c h l o r i d e . The p r e c i p i ­

t a t e tha t fo rmed i n i t i a l l y , d i s s o l v e d m o r e or l e s s r a p i d l y

depend ing on the quan t i t y of m y r i s t o y l c h l o r i d e added . Af­

t e r s t a n d i n g 20 h o u r s at room t e m p e r a t u r e , 3 /4 of the

p y r i d i n e was r e m o v e d by d i s t i l l a t i o n in a vacuum and the

r e s u l t i n g s o l u t i o n was p o u r e d with s t i r r i n g in to a four m o ­

l a r e x c e s s of p o t a s s i u m c a r b o n a t e (5 p e r c e n t s o l u t i o n ) b a ­

sed on the h y d r o c h l o r i c ac id f o r m e d . An equal vo lume of

n - b u t a n o l was then added and the e s t e r s of s u c r o s e w e r e

p u r i f i e d of the e x c e s s s u c r o s e by five e x t r a c t i o n s of the

bu t ano l p h a s e with equal v o l u m e s of 5 p e r c e n t aqueous s o ­

dium c h l o r i d e . T h e s e aqueous p h a s e s w e r e in t u r n e x t r a c ­

t ed wi th a s econd vo lume of n - b u t a n o l . The combined o r ­

gan i c p h a s e s w e r e d r i e d with anhydrous sodium s u l f a t e ,

f i l t e r e d , and the m y r i s t a t e e s t e r s ob ta ined upon vacuum

d i s t i l l a t i o n of the bu t ano l and the r e m a i n i n g p y r i d i n e .

4 . _A_naly_tical__Meth_o_ds_

a. Sapon i f i c a t i on and F a t t y Acid Content D e t e r m i ­

n a t i o n . - F o r the s a p o n i f i c a t i o n of the s u c r o s e e s t e r s , the

m i c r o method of M a t t h e r and Z i e g e n s p e c k ( 6 3 ) and the s e m i -

- 3 4 -

micro method of Mi tche l l , Smith, and Money(64) could not

be used for solubi l i ty r e a s o n s . The saponificat ion p r o c e ­

dure employed throughout the p resen t study was developed

for the sucrose e s t e r s . It compr ises a minimum of hand­

l ing, r e q u i r e s only 0.5 m . e . of sucrose e s t e r and is ac ­

cura te to be t t e r than one pe rcen t .

The dr ied sucrose fatty e s t e r (0 .3 to 0.5 m . e . )

samples were weighed into 150 ml . p r e s s u r e bot t les and

exactly 25 ml . of 75 percent aqueous ethanol containing

1 m . e . of potass ium hydroxide was added. The bot t les o o

were then heated in a water bath at 80 to 90 C. for ap­

proximate ly two h o u r s . The excess alkal i was t i t r a t ed

with 0.05 N hydrochlor ic acid to a phenolphthalein end

po in t . The saponificat ion equivalents were ca lcula ted in

the usual manner using the difference between the t i t r a t i on

of the blanks and that of the runs .

For the fatty acid content de te rmina t ion , the so­

lut ions from the saponification de terminat ions were t r a n s ­

fe r r ed into 250 ml . round bottom f l a sks , using ethanol to

wash the contents quant i ta t ive ly . The ethanol was then

removed by d i s t i l l a t ion and the fatty acid p rec ip i t a t ed by

the addition of 100 ml . of cold water and 2 to 3 ml . of 2

N hydrochlor ic acid . After cooling, the fatty acid p r e c i -

- 3 5 -

p i t a t e was f i l t e red and washed with cold water unt i l free

of chlor ide ions . The fatty acid on the funnel could be

weighed (using a c in te red glass funnel) and t i t r a t e d in

ethanol solution to the bromothymol blue end point (pH 6

to 7.6) with 0.05 N sodium hydroxide . Blanks, using the

same volume of ethanol , were subt rac ted from the values

obtained for these t i t r a t i o n s . The s tandard base and acid

used in the above procedures were p repared from carbon

dioxide free water and s tandardized against potassium hy­

drogen ph tha la te .

b . Formyl Group De te rmina t ion . - A procedure was

developed during the course of this work to analyze the su­

c rose fatty e s t e r s for formyl g roups . When known amounts

of sodium formate were reduced with magnesium and hydro­

ch lor ic acid, the co lo r ime t r i c determinat ion of the formal ­

dehyde produced gave a measure of the sodium formate o r i ­

ginally p r e s e n t . This was best accomplished by d i s t i l l ing

pa r t of the react ion mixture and doing the formaldehyde e s ­

t imat ion on the d i s t i l l a t e . This p rocedure , which is a mo­

dif icat ion of the method of W.M. Grant (65), was found to

be eas i e r to reproduce than the l a t t e r .

The method was s tandardized as fol lows. The so­

dium formate (between 0.05 and 0.7 mM) was dissolved in

-36-

in a 100 m l . round bottom flask using ten ml . of w a t e r .

Concentra ted hydrochlor ic acid (half an m l . ) was added to

d i sp lace the carbon dioxide and the flask was immersed in

an ice ba th . Magnesium meta l , 0.6 g . , ( 3 0 " 1/8" 1/5000")

was added, followed by 4.5 ml . of concentra ted hydrochlo­

r i c acid added in 0.5 ml . al iquots at two minute i n t e r v a l s .

After completion of the r eac t ion , ten ml . of water was added

and ten ml . of the solution was d i s t i l l ed into a ten ml . vo­

lume t r i c f lask . For the co lo r ime t r i c de te rmina t ion , one

m l . a l iquots of the d i s t i l l a t e (or diluted solutions of the

d i s t i l l a t e ) were t r ea t ed with ten ml . of chromotropic acid

reagent (66) . The percent t r ansmis s ion was measured at

570 myi\ in a Coleman Junior spect rophotometer using 19X

150 mm. cuve t t e s . Ery th r i to l was oxidized with per ioda te

and the formaldehyde produced (two moles per mole of gly­

col) was used to s tandard ize the co lo r ime t r i c method (66) .

The express ion obtained was; number of mi l l imoles of for­

maldehyde in a c u v e t t e , log 1 /T . The r e s u l t s of the con-2155

t r o l run with sodium formate are given in F i g . 1 as a plot

of the mi l l imoles of formaldehyde d i s t i l l ed against m i l l i ­

moles of sodium formate reduced .

The reduction of the sucrose m y r i s t a t e s by the

above procedure proceeded abnormally slow due to the poor

solubi l i ty of the e s t e r s in the media . It became necessa ry

3 7 -

0.10^.

0.08

0 .06 . .

0 .04 . .

0 . 0 2 . .

0.2 0.4 0.6 M i l l i m o l e s of Sodium F o r m a t e Reduced

F i g . 1. S t a n d a r d Curve for the F o r m y l Group D e t e r m i n a t i o n P r o c e d u r e .

- 3 8 -

t he re fo re to hydrolyze the e s t e r s of the fatty acid before

ca r ry ing out the r educ t ions . This was done by t r ea t ing

the aqueous solution of the es te r (0 .5 g. in 20 m l . ) with P

four m l . of ten percent sodium hydroxide at 100 C. for 30

minu te s . The fatty acid was then p rec ip i t a ted by adding

five ml . of ten percent magnesium chlor ide solut ion. To

fac i l i t a t e the f i l t ra t ion of the magnesium m y r i s t a t e , 2 N

hydrochlor ic acid was added to dissolve the magnesium hy­

droxide p r ec ip i t a t e that had formed when the magnesium

chlor ide was added. Phenolphthalein was added to make

sure that the solution remained b a s i c . The f i l t r a t e was

col lected in a 100 ml . round bottom flask, evaporated to

dryness and the formyl content of the res idue was de te r ­

mined as descr ibed for sodium formate .

Two control runs , s imulat ing sucrose myr i s t a t e

s ample s , were made to tes t this p rocedu re . Run A con

ta ined 0.32.44 g. of suc rose , 0.1506 g. of m y r i s t i c acid

and 0.011047 g. of sodium formate- Run B contained

0.4565 g. of suc rose , 0.1814 g. of myr i s t i c acid and

0.059629 g. of sodium fo rmate . The r ecove r i e s of formal ­

dehyde were 66 and 61 percent of the amounts expected on

the bas i s of the r e s u l t s plotted in F i g . 1.

c . Paper Chromatography of the Sucrose E s t e r s . -

-39-

For this work, 8x22 inch sheets of Whatman paper No. 1

were used and the descending method was employed exclu­

s ive ly . The compounds chromatographed were added to

the paper as one applicat ion of a four percent solution on

points spaced 1.25 inches apar t along the s t a r t ing l ine ,

which was p a r a l l e l to and 4.5 inches from the narrow edge

of the pape r . One applicat ion of a four percent solution

approximately two cm. in diameter r ep re sen t s 200 micro

grams of compound. After the applied spots had dr ied , the

paper was placed in a chromatographic cabinet sa tu ra ted

with the bottom phase of the solvent system employed. The

top phase was then added to the solvent trough and the

front was allowed to move down 12 to 13 inches below the

s t a r t i ng l i n e . The paper was next dr ied for the spray ing .

n-Butanol sa tu ra ted with wate r , methyl ethyl ke­

tone sa tu ra ted with water and n-butanol : ethanol: water

(5:1:4) were the most frequently used solvent s y s t e m s .

A number of spray reagents have been invest igated for use

with the fatty e s t e r s of sucrose on paper chromatograms .

The hydroxamic acid spray reagent for detect ing e s t e r s on

paper chromatogr ams was t r i ed without success (67).

The following th ree reagents have found useful applicat ion

during this work.

- 4 0 -

The a n i l i n e p h o s p h a t e s p r a y used was a m o d i f i c a ­

t i on of the r e a g e n t d e s c r i b e d by Bryson and M i t c h e l l ( 6 8 ) .

The r e a g e n t was p r e p a r e d by mix ing b e f o r e u s e , two vo lu ­

m e s of 2 N aqueous p h o s p h o r i c ac id and one volume of 2 N

a n i l i n e in e t h a n o l , fol lowed by t h r e e vo lumes of g l a c i a l a c e ­

t i c ac id to d i s s o l v e the s a l t p r e c i p i t a t e . The d r i e d d e v e ­

loped c h r o m a t o g r a m s w e r e s p r a y e d h e a v i l y with the s o l u t i o n ,

o

a l lowed to d ry and then h e a t e d at 105 C. for 10 to 20 m i n u ­

t e s . Any r e d u c i n g p e n t o s e s and h e x o s e s w e r e d e t e c t e d as

brown s p o t s a long with d i s a c c h a r i d e s ( such as s u c r o s e )

which a r e h y d r o l y z e d by the r e a g e n t to h e x o s e s .

The g lyco l s p r a y r e a g e n t of Lemieux and Bauer

(69) was p r e p a r e d by mix ing be fo re u s e , one vo lume of one

p e r c e n t p o t a s s i u m p e r m a n g a n a t e in two p e r c e n t sodium c a r ­

b o n a t e and four vo lumes of two p e r c e n t aqueous sodium p e ­

r i o d a t e . The d r i e d deve loped p a p e r c h r o m a t o g r a m s w e r e

s p r a y e d h e a v i l y with the so lu t i on and p r e f e r a b l y p l a c e d in

a humid a t m o s p h e r e du r ing the induc t ion t i m e . Glyco ls

a p p e a r e d as ye l low s p o t s on a p u r p l e b a c k g r o u n d . Wash ing

the e x c e s s p e r m a n g a n a t e - p e r i o d a t e off the p a p e r r e s u l t e d

in a p e r m a n e n t brown spot on a whi te b a c k g r o u n d .

The a n i l i n e h y d r o g e n p h t h a l a t e (70) s p r a y r e a g e n t

was p r e p a r e d by d i s s o l v i n g 930 m g . of a n i l i n e and 1.6 g .

- 4 1 -

of phthal ic acid into 100 ml . of n-butanol sa tu ra ted with

wa t e r . The d r ied , developed, paper chromatogr ams were o

sprayed with the reagent solution and heated at 105 C. for

10 to 20 minu tes . This reagent is s imi l a r in se lec t iv i ty

to the anil ine phosphate sp ray .

Although the sucrose fatty e s t e r s show much

s t reak ing on paper chr omatograms, this technique has p ro ­

ven useful in obtaining qual i ta t ive information on a sucrose

e s t e r s ample . Of the solvent system employed, n -bu tanol /

water was found to give good reso lu t ions and was eas i e r to

use than the o t h e r s . With this solvent the Rf values of su

c r o s e , g lucose , f ructose and sucrose monomyris ta te were

found to be 0 .045, 0 .075, 0.10 and 0 .75 , r e spec t ive ly .

Aniline phosphate was the p re f e r r ed reagent for the fatty

e s t e r s of s u c r o s e . It had a higher sens i t iv i ty than the ani­

l ine phthalate reagent and gave chr omatograms eas ie r to

read than those sprayed with the permanganate-p er iodate

r e a g e n t .

A mixture of sucrose mono- and d imyr i s t a t e s was

not separa ted on paper with n-butanol /water . Both com­

pounds were found between R£ values of 0.7 to 0 . 8 5 . How­

ever , during the spraying with anil ine phosphate , the poly-

m y r i s t a t e s of sucrose were not wetted by the reagent while

- 4 2 -

the monoes te r s w e r e . Examination during the spraying

the re fo re gave information regard ing the composition of

the mixture chromatographed .

d. Par t i t ion Chromatography on C e l i t e . - The p r e ­

pa ra t ive pa r t i t ion chromatography of carbohydrates on Ce­

l i t e columns has recent ly been the subject of a publicat ion

(71) . The method used for the sucrose e s t e r s was essen­

t ia l ly the s a m e . The Cel i te 535 employed as the absorbent

was t r ea t ed with concentra ted hydrochlor ic acid before use

(71) . The columns were cons t ruc ted by f i r s t , wetting the

Cel i te with the water phase , using a one to one volume to

weight r a t i o , s lu r ry ing this mixture in the developing

phase and compress ing into a chromatographic tube with

a close fi t t ing p lunger . An approximately ten percent so­

lution of the sample (preferably in the developing phase)

was absorbed on dry Cel i te (1 ml . per g. ) and the r e s u l ­

ting powder was packed to the top of the column which was

jus t f i l led with developing phase . The top surface was p r o ­

tec ted by a f i l t e r pape r . The developing phase was then

pe rco la ted through the column by gravi ty and the eluate

was f rac t ionated by an automatic co l l ec to r . When two or

more i r r i g a t i n g solvents were used in success ion , the

level of the previous solvent was just below the top of the

column before the next solvent was added.

- 4 3 -

Since a l l the compounds c h r o m a t o g r a p h e d c o n t a i ­

ned s u c r o s e and s u c r o s e d e r i v a t i v e s , the f r a c t i o n s w e r e

t e s t e d for t h e i r s u c r o s e con ten t u s ing the a n t h r o n e r e a g e n t

( 7 2 ) . Th i s r e a g e n t does not funct ion p r o p e r l y in the p r e ­

s e n c e of a l c o h o l s and k e t o n e s , and i t was t h e r e f o r e n e c e s ­

s a r y to f r e e the s a m p l e s of o r g a n i c s o l v e n t s b e f o r e add ing

the r e a g e n t . T h i s was c o n v e n i e n t l y done by p l a c i n g the

c u v e t t e (19x150 m m . ) of a Coleman Junior s p e c t r o p h o t o m e ­

t e r , c o n t a i n i n g the a l iquo t to be a n a l y z e d , in a t e s t tube

i m m e r s e d in a ba th of oil h e a t e d to about 90 C. The bath

was then p l a c e d in a l a r g e vacuum d e s s i c a t o r for e v a p o r a ­

t ion in vacu_o_. T h r e e m l . of w a t e r and s ix m l . of a n t h r o n e

r e a g e n t w e r e then a d d e d . It i s to be noted tha t un i fo rm

and r a p i d add i t i on of the a n t h r o n e r e a g e n t is n e c e s s a r y and

t h i s is b e s t a c c o m p l i s h e d with a s y r i n g e p i p e t t e . The an ­

t h r o n e r e a g e n t was p r e p a r e d by d i s s o l v i n g two g . of a n t h r o ­

ne in a l i t e r of c o n c e n t r a t e d s u l f u r i c ac id ( 7 2 ) . The a n t h r o ­

ne used was o b t a i n e d from the r e d u c t i o n of a n t h r a q u i n o n e

wi th t in and h y d r o c h l o r i c ac id ( 7 3 ) .

Af ter s t a n d i n g at room t e m p e r a t u r e for one h o u r ,

t he g r e e n coloul* p r o d u c e d was r e a d at 620 myc\ . S u c r o s e

was u sed to s t a n d a r d i z e the m e t h o d . A t y p i c a l run with

s u c r o s e i s g iven in T a b l e I . The s u c r o s e con ten t of a tu

be was c a l c u l a t e d from t h i s s lope u s ing the e x p r e s s i o n ;

44

m i c r o g r a m s of s u c r o s e ^ , l o g 1/T

"* 7 . 5 5 5 x 10"*3

T a b l e I

S t a n d a r d i z a t i o n of t h e A n t h r o n e M e t h o d f o r t h e D e t e r m i n a ­

t i o n of S u c r o s e C o n t e n t s .

M i c r o g r a m s of s u c r o s e in c u v e t t e

18 .7

37 . 4

39 .8

69 .3

7 4 . 8

149 .7

L o g 1 /

0 . 1 4 4

0 . 2 8 2

0 . 3 0 4

0 . 5 1 8

0 . 5 6 4

1 . I l l

T

A v e r a g e

S l o p e

7 . 7 0 x l 0 ~ 3

7 . 5 4 "

7 . 6 5 "

7 . 4 8 "

7 . 5 4 "

7 . 4 2 "

7 . 5 5 5 x l 0 ~ 3

When t h e d e v e l o p i n g p h a s e had no t c o m p l e t e l y r e ­

m o v e d t h e c o m p o n e n t s f r o m t h e c o l u m n , t h e l a t t e r w a s ex

t r u d e d and t h e p o s i t i o n s of t h e b a n d s w e r e d e t e r m i n e d by

s p r a y i n g t h e c o l u m n t h r o u g h a m a s k ( 7 4 ) w i t h a one p e r ­

c e n t s o l u t i o n of p o t a s s i u m p e r m a n g a n a t e in 2 . 5 N s o d i u m

h y d r o x i d e ( 7 5 ) .

A t t e m p t s w e r e m a d e to a n a l y z e t h e r e a c t i o n m i x ­

t u r e s of t h e p r e p a r a t i o n of s u c r o s e m y r i s t a t e by t r a n s e s

t e r i f i c a t i o n u s i n g C e l i t e p a r t i t i o n c h r o m a t o g r a p h y . P r e -

- 4 5 -

l i m i n a r y s t u d i e s with the so lven t s y s t e m s n - b u t a n o l s a t u ­

r a t e d wi th w a t e r and m e t h y l e thyl ke tone s a t u r a t e d with

w a t e r showed tha t t h e s e s o l v e n t s e lu ted the e s t e r s a m p l e s

too r a p i d l y . O the r c h r o m a t o g r ams with the so lven t s y s t e m

h e p t a n e : m e t h a n o l : w a t e r (15 :9 :1 ) showed tha t the hep t ane

p h a s e was e lu t i ng some p o l y e s t e r s of s u c r o s e s i n c e the

e l u a t e c o n t a i n e d a s m a l l c a r b o h y d r a t e b a n d . However no

s e p a r a t i o n of the mono- and d i e s t e r s was a c h i e v e d .

A c h r o m a t o g r a p h i c p r o c e d u r e was deve loped in

which the co lumns w e r e e lu t ed with t h r e e c o n s e c u t i v e s o l ­

v e n t s , n a m e l y , with h e p t a n e s a t u r a t e d with w a t e r , then

wi th n - b u t a n o l , and, f i na l ly with w a t e r . The co lumns

w e r e p r e p a r e d as a s l u r r y with n - b u t a n o l s a t u r a t e d with

w a t e r and then i r r i g a t e d with h e p t a n e p h a s e to r e m o v e the

b u t a n o l . Two C e l i t e co lumns (25 g . of C e l i t e in 26 m m .

t u b e s ) w e r e p r e p a r e d in t h i s way to ana lyze a m i x t u r e

(25 m g . ) of s u c r o s e , s u c r o s e m o n o m y r i s t a t e s and of s u c r o ­

se p o l y m y r i s t a t e s . The co lumns w e r e deve loped f i r s t with

125 m l . of h e p t a n e s a t u r a t e d with w a t e r , then with 100 m l .

of n - b u t a n o l and f ina l ly with 100 m l . of w a t e r . The e l u a -

t e s w e r e c o l l e c t e d s e p a r a t e l y and a n a l y z e d for t h e i r s u c r o ­

se con ten t wi th a n t h r o n e . The r e c o v e r i e s w e r e 9 3 . 5 p e r ­

c e n t . The r e s u l t s i n d i c a t e d the p r e s e n c e of 1 7 1 9 % of the

s u c r o s e in the h e p t a n e f r a c t i o n , 61-64% of the s u c r o s e in

- 4 6 -

the bu t ano l f r a c t i o n and 20% of the s u c r o s e in the w a t e r

f r a c t i o n .

The c o u r s e of a t r a n s e s t e r i f i c a t i o n r e a c t i o n was

fo l lowed u s ing the C e l i t e c h r o m a t o g r a p h i c method to ana ­

l y z e the r e a c t i o n m i x t u r e s i s o l a t e d a f t e r a v a r i e t y of r e ­

a c t i o n t i m e s . S u c r o s e (75 mM) and me thy l m y r i s t a t e

(25 mM) w e r e r e a c t e d with 2 . 5 m i l l i m o l e s of p o t a s s i u m

c a r b o n a t e in 86 m l . of DMF as d e s c r i b e d on page 3 1 . The

p r e s s u r e was kept at 49 m m . and the t e m p e r a t u r e of the

© o

r e f l u x i n g s o l u t i o n was c o n s t a n t be tween 76 and 77 C. The

s o l u t i o n was s a m p l e d from t i m e to t ime th rough a s a m ­

p l i n g tube wi thout d i s r u p t i n g the r e a c t i o n c o n d i t i o n s . A

0 . 1 m l . vo lume of the r e a c t i o n m i x t u r e was used for the

c h r o m a t o g r a p h i c s e p a r a t i o n of the componen t s on C e l i t e

c o l u m n s , 25 g . , 26 m m . in d i a m e t e r . The c a r b o h y d r a t e

c o n t e n t s of the t h r e e f r a c t i o n s were d e t e r m i n e d with the

a n t h r o n e r e a g e n t . The exac t amount of s u c r o s e in the a l i ­

quots be ing unknown, the r e s u l t s given in Tab le II a p p e a r

as the p e r c e n t a g e s u c r o s e in a given f r a c t i o n to the t o t a l

amount of s u c r o s e found in a l l t h r e e f r a c t i o n s . The f i r s t

f r a c t i o n c o n t a i n e d only t r a c e s ( l e s s than 1%) of s u c r o s e

d e r i v a t i v e and is not r e p o r t e d in F i g . 2 .

-47-

T a b l e II

C h r o m a t o g r a p h i c A n a l y s e s of the R e a c t i o n P r o d u c t s from

the T r a n s e s t e r i f i c a t i o n of Methyl M y r i s t a t e with S u c r o s e

T i m e in h o u r s

0

0 . 5

1 . 0

3 . 3

5 . 3

8 .3

1 0 . 6

H e p t a n e f r a c t i o n

0

0

0 . 17

0

0

0

0 . 3 5

P e r c e n t S u c r o s e n - B u t a n o l f r a c t i o n

0 . 6

11 .2

1 7 . 3

31 .2

3 9 . 9

3 4 . 0

39 . 1

W a t e r f r a c t i o n

9 9 . 4

88 .2

8 2 . 7

6 8 . 8

6 0 . 1

66 . 0

6 0 . 5 5

5 . D e t e r m i n a t i o n of the Number of M y r i s t o y l Groups O c ­

cupying the 6 and 6 ' - P o s i t i o n s in a S u c r o s e M y r i s t a t e .

a . The P r e p a r a t i o n of the Tosy l E s t e r s ( 7 6 ) . -

The c a r b o h y d r a t e s a m p l e to be t o s y l a t e d was weighed in to

an oven d r i e d g round s t o p p e r e d f l a sk and d i s s o l v e d with

d ry p y r i d i n e . The l eve l of the p y r i d i n e employed was b a ­

sed on the t o s y l c h l o r i d e u s e d , 100 m l . of p y r i d i n e p e r

25 g . of t o s y l c h l o r i d e . T h i s so lu t i on was cooled in an

i c e ba th and a 10 to 25 p e r c e n t e x c e s s of t o s y l c h l o r i d e

was a d d e d . The s o l u t i o n was wel l s t o p p e r e d and kept at

48 100

80..

6 0 . . CO

o n o

+J

a> u

£ 40

20.-

•+•

- G

— O

(1) . Water E lua te .

(2 ) . Butanol Eluate

•4- -f 4 6

Time in Hours 8 10

F i g . 2 . Kinetic Run of the Transe? t e r i f i ca t ion Reaction

- 4 9 -

5 C. for ten to e l even d a y s . After t h i s t i m e , the p y r i d i -

n ium h y d r o c h l o r i d e fo rmed was r e m o v e d by f i l t r a t i o n and

washed once with some p y r i d i n e . The e x c e s s t o sy l c h l o r i ­

de in the p y r i d i n e so lu t i on was d e s t r o y e d by the slow a d d i ­

t i o n , with s h a k i n g , of w a t e r equ iva l en t to about half of

the t o s y l a t i n g agent o r i g i n a l l y u s e d . The so lu t i on was then

e v a p o r a t e d to n e a r d r y n e s s in a vacuum and the r e s i d u e d i s ­

so lved in c h l o r o f o r m (100 m l . of c h l o r o f o r m p e r 25 g . of

p r o d u c t ) . The c h l o r o f o r m so lu t ion was e x t r a c t e d twice wi th

10 p e r c e n t s u l p h u r i c a c i d , once with s a t u r a t e d sodium b i ­

c a r b o n a t e s o l u t i o n , washed with w a t e r , and d r i e d with an­

h y d r o u s sodium s u l f a t e . The t o sy l e s t e r was ob ta ined by

e v a p o r a t i n g the f i l t e r e d c h l o r o f o r m so lu t i on to d r y n e s s .

The su l fur c o n t e n t s of the t o s y l e s t e r s was d e t e r ­

mined by the method of Sundberg and Roger ( 7 7 ) . The e s ­

t e r ( 1 0 to 40 m g . ) was b u r n e d in an oxygen a t m o s p h e r e

and the g a s e s w e r e c o l l e c t e d in a Gro te a b s o r b e r with

t h r e e p e r c e n t n e u t r a l i z e d hydrogen p e r o x i d e . The s o l u ­

t ion was t r a n s f e r r e d to a 125 m l . E r l e n m e y e r , bo i l ed to

expel the c a r b o n d i o x i d e , and the s u l f u r i c ac id was t i t r a ­

t ed with 0 .05 N sodium h y d r o x i d e to the me thy l r e d end

p o i n t . The su l fur ana ly se s of 1, 2-3 , 5 - d i - O - m e t h y l e n e

O 0

6 - O - t o s y l - D - g l u c o f u r a n o s e ( m . p . 112 .5 -113 C . ) by the

above method gave v a l u e s of 8 . 9 1 , 8 . 8 2 , 8 . 9 2 , 8 .96 and

- 5 0 -

8 .97 p e r c e n t s u l f u r . The c a l c u l a t e d va lue i s 8 .95 p e r c e n t

s u l f u r .

The r e s u l t s of the t o s y l a t i o n s and su l fur a n a l y s e s

a r e r e p o r t e d in T a b l e VI I .

b . The Iod ina t i on of Tosy l E s t e r s ( 7 6 ) . In a l l

e x p e r i m e n t s c a r r i e d out , the t o sy l e s t e r was weighed i n ­

to a 125 m l . p r e s s u r e b o t t l e and d i s s o l v e d with a ten p e r ­

cen t s o l u t i o n of sodium iod ide in a c e t o n e . Th i s r e a g e n t

( F i n k e l s t e i n 1 s r e a g e n t ) was p r e p a r e d by d i s s o l v i n g ten

g r a m s of sodium iod ide in 100 m l . of a c e t o n e . Between

12 and 14 m l . of the sodium iod ide s o l u t i o n w e r e u sed p e r

g r a m of e s t e r , t h i s amount be ing at l e a s t a twofold e x c e s s

of sodium iod ide over the t o sy l compound . The s e a l e d bo t ­

t l e was then h e a t e d in s t eam for the l eng th of the r e a c t i o n

t i m e . After c o o l i n g , the b o t t l e was opened and the a c e t o ­

ne e v a p o r a t e d . Equal vo lumes of benzene and w a t e r w e r e

added to the r e s i d u e u s ing 100 m l . of the so lven t m i x t u r e

for one to t h r e e g r a m s of t o s y l e s t e r o r i g i n a l l y e m p l o y e d .

A l i t t l e sodium t h i o s u l f a t e was added at t h i s s t a g e to r e ­

duce any f r ee iod ine p r e s e n t . The benzene l a y e r was then

washed once with w a t e r , d r i e d with anhydrous sodium sul

f a t e and the p r o d u c t was ob ta ined by e v a p o r a t i o n of the

benzene s o l u t i o n to d r y n e s s .

- 5 1 -

The ex t en t of r e p l a c e m e n t of the t o sy loxy group

was ob t a ined from the iod ine con ten t of the iodo d e r i v a t i

v e s . The s a m p l e to be a n a l y z e d (10 to 40 mgm . ) was bur

ned in an oxygen a t m o s p h e r e and the l i b e r a t e d iod ine c o l ­

l e c t e d in a Gro t e a b s o r b e r with five p e r c e n t sodium h y d r o ­

x ide ( 7 7 ) . The c o n t e n t s of the a b s o r b e r w e r e t r a n s f e r r e d

to a sodium a c e t a t e , a c e t i c ac id buffer and b r o m i n e was

added to ox id ize a l l the iod ine a b s o r b e d to i o d a t e . After

d e s t r o y i n g the e x c e s s b r o m i n e , the ioda te was t i t r a t e d in

the u s u a l way with t h i o s u l f a t e . The iod ine ana lyses of

1, 2 - 3 , 5 - d i - 0 - m e t h y l e n e 6 - d e o x y i o d o - D - g l u c o f u r a n o s e

( m . p . 9 6 . 5 ° C . ) gave v a l u e s of 4 1 . 8 5 , 4 0 . 1 3 , 3 9 . 9 1 , 4 0 . 2 9 ,

4 0 . 1 8 and 4 0 . 8 0 p e r c e n t i o d i n e . The c a l c u l a t e d va lue is

4 0 . 4 2 p e r c e n t i o d i n e .

The r e s u l t s of the i o d i n a t i o n e x p e r i m e n t s a r e g i ­

ven in T a b l e VII .

6 . Sodium P e r i o d a t e Ox ida t ions (78)

The s u g a r ( 0 . 2 mM for h e x o s e s and 0 .1 mM for

d isac cha r i d e s ) was weighed and d i s s o l v e d in a 100 m l .

v o l u m e t r i c f l ask with 75 m l . of w a t e r . At t e m p e r a t u r e

e q u i l i b r i u m , 20 m l . of 0 . 1 M sodium m e t a - p e r i o d a t e w e r e

p i p e t t e d in to the s u g a r s o l u t i o n and the vo lume was made

up to the m a r k . To s top the r e a c t i o n , 10 m l . a l i q u o t s of

- 5 2 -

the ox ida t ion m i x t u r e was p i p e t t e d in to a known e x c e s s of

sod ium a r s e n i t e (10 m l . of 0 .05 N sodium a r s e n i t e ) con­

t a i n i n g an e x c e s s of sodium b i c a r b o n a t e and a few p o t a s s i u m

iod ide c r y s t a l s . After ten m i n u t e s , the e x c e s s a r s e n i t e

was t i t r a t e d with 0 .02 N iod ine so lu t ion to a s t a r c h end

p o i n t . The d i f f e r e n c e be tween th i s t i t e r and tha t of a r e a ­

gent b lank was t aken as a m e a s u r e of the ex ten t of o x i d a ­

t i o n . T a b l e III l i s t s the v a l u e s ob ta ined when g l u c o s e and

o s u c r o s e w e r e ox id i zed at 2 4 . 8 C. The pH of the b lank

run was 5 .2 whi le t ha t of the s u c r o s e ox ida t ion m i x t u r e

a f t e r two days was 3 . 8 . The up take of oxidant shows tha t

no h y d r o l y s i s of the g l y c o s i d i c bonds o c c u r r e d .

T a b l e III

The Oxida t ion of S u c r o s e and Glucose with Sodium meta

p e r i o d a t e .

S u c r o s e T i m e mM p e r i o d a t e

in h o u r s p e r m M .

0 . 3

0 . 8

3

4

1.65

2 . 2 8

2 . 7 0

2 . 8 5

Glucose T i m e mM p e r i o d a t e

in h o u r s p e r mM .

0 . 3

0 .8

3

3 .38

4 . 6 3

4 .96

48 3 . 0 1

- 5 3 -

The r e s u l t s of the oxidation of sucrose monomy­

r i s t a t e are repor ted in Table IX.

7. Reduction of Tosyl Es t e r s with Lithium Aluminum Hy­

dr ide .

For the reduct ions ca r r i ed out in diethyl e the r ,

the tosyl e s t e r was added, as a concentra ted solution in

dry benzene, to a five equivalent excess of l i thium a lumi­

num hydride s l u r r i e d in e t h e r . The reac t ion mix tu re , s t i r ­

red through a mercury sea l , was refluxed for 48 h o u r s . The

excess reagent was destroyed f i r s t by the addition of a l i t ­

t le water followed by the addition of a la rge excess of wa­

t e r . The water phase from the f i l t r a t e of the above mix­

tu re was ex t rac ted with benzene to remove any p -d i to ly l -

disulf ide (79) . The aqueous solution was then passed th rou­

gh a column of 200-400 mesh Dowex 1-X10 (qua te rnary am­

monium type anion exchange res in ) and then through a co­

lumn of Amber l i te IRC50(H) (a carboxyl ic type r e s in ma­

nufactured by Rohm and Hass C o . ) . The eluate i ssuing

from the cation exchange r e s in always had a pH of 3.9

and boiling did not change this pH . Sufficient sodium b i ­

carbonate was added to r a i s e the pH to 7 before the eva­

pora t ion of the wa te r . The concent ra te was then freed of

the ions added by pass ing it through a smal l excess of the

- 5 4 -

two r e s i n s m e n t i o n e d a b o v e . The p r o d u c t was ob t a ined

f rom t h i s s o l u t i o n by e v a p o r a t i o n of the w a t e r to d r y n e s s .

The r e d u c t i o n in t e t r a h y d r ofur an was c a r r i e d out

a c c o r d i n g to the p r o c e d u r e d e s c r i b e d for d i e thy l e t h e r .

The t o s y l e s t e r was d i s s o l v e d in t e t r a h y d r o f u r a n i n s t e a d

of b e n z e n e for the add i t i on to the l i t h i u m a luminum h y d r i d e .

An h o m o g e n e o u s r e a c t i o n m i x t u r e was ob t a ined with t h i s

s o l v e n t and a s h o r t e r r e a c t i o n t ime of 20 h o u r s was e m ­

p loyed .

F o r the d e t e r m i n a t i o n , as a c e t i c a c i d , of the

«(-hydroxyethyl g roups p r e s e n t in l i t h i u m a luminum h y d r i d e

r e d u c t i o n p r o d u c t s , the p r o c e d u r e of Lemieux and P u r v e s

(80) was e m p l o y e d . F o r t h i s a n a l y s i s , the s a m p l e was

o x i d i z e d in 30 p e r c e n t aqueous c h r o m i u m t r i o x i d e and the

a c e t i c ac id fo rmed was d i s t i l l e d and t i t r a t e d . A p r o v i s i o n

i s i nc luded in the method for the c h r o m i c ac id which may

p a s s in to the d i s t i l l a t e . S u c r o s e o c t a a c e t a t e ( m . p . 85 -

89 C . ,|»l<Ljh60 . 5 ) was a n a l y z e d by t h i s p r o c e d u r e and gave

an e q u i v a l e n t weight of 8 5 . 4 g r a m s . T h e t h e o r e t i c a l va lue

i s 8 4 . 8 g r a m s . Th i s r e p r e s e n t s a r e c o v e r y of a c e t i c ac id

of b e t t e r than 99 p e r c e n t .

The r e s u l t s of the r e d u c t i o n of s u c r o s e t o s y l a t e

and of s u c r o s e m o n o m y r i s t a t e t o s y l a t e a r e g iven in T a b l e X.

- 5 5 -

8. The Prepara t ion of Radioactive Sucrose Pa lmi ta tes and

the Chromatographic Separat ion of a Mixture of Sucrose

Aceta tes .

During the course of this work, two samples of

rad ioac t ive sucrose pa lmi ta tes were p repared at the r e ­

quest of the Sugar Research Foundation for use by Dr.

Quaste l of McGill Un ive r s i ty . His s tudies of the metabo­

l ism of these e s t e r s have shown that the molecules are

hydrolyzed in the stomach and consequently do not enter in

the blood s t r e a m . Also, a sample of sucrose monoacetate

was submit ted to us for s t r u c t u r a l study by Dr . K.M. Her-

s te in of the Hers te in Labora to r i e s of New York. The sam­

ple was descr ibed by the sender as a pure sucrose mono-

a c e t a t e . An account of this work is given below to p r e ­

se rve the continuity of the following sec t ion .

a. The Prepara t ion of Radioactive Sucrose Mono-

p a l m i t a t e s . - The rad ioac t ive pa lmi t ic acid (27.9 mg.

1 4 ac t iv i ty : 0.1 m i l l i c u r i e , C at carbon 1) was dissolved

in 20 ml . of dry benzene and t r ea t ed with an e ther solution

of diazomethane . The diazomethane was p repared from

N-methyl N-n i t roso urea by the method of Arndt (82) . After

the methyla t ion , the solvents were removed and the r a d i o ­

act ive methyl pa lmi ta te was t r a n s f e r r e d to a ten ml . volu

m e t r i c flask as a benzene solu t ion .

-56-

The two p repa ra t ions were made according to the

t r a n s e s t e r i f i c a t i o n procedure given on page 3 1 . For the

f i r s t p r e p a r a t i o n , the rad ioac t ive methyl pa lmi ta te i s o l a ­

ted from two m l . of the stock solution was diluted with

five grams of cold methyl pa lmi ta te and t rans es te r i f i ed for

eleven h o u r s . The nine gram yield i so la ted with n-butanol

had an act iv i ty of two mic rocu r i e s per g ram. For the s e ­

cond p r epa ra t i on , eight ml . of the radioact ive methyl pa l ­

mi ta te were di luted with 1.275 grams of cold methyl e s t e r .

The rad ioac t ive e s t e r i so la ted (1.925 g . ) had a calculated

ac t iv i ty of 40 m i c r o c u r i e s per gram of e s t e r .

b . The Analyses of a Mixture of Sucrose Ace t a t e s . -

The sucrose ace ta te sent by Dr . Hers te in was or ig inal ly

obtained from the reac t ion of one mole of sucrose with one

mole of ace t ic anhydride in pyr idine followed by p rec ip i

ta t ion of the product from n-butanol . This amorphous so­ts o

lid mel ted in the range of 57 to 70 C. and had a ro t a t ion ,

l^L+54.2 (in w a t e r ) . The e s t e r saponified at 260 grams

but gave acetyl group equivalents of 382 and 383 grams

when analyzed for the acetyl content . The theore t i ca l va­

lue for the acetyl group equivalent of sucrose monoacetate

is 384 g r a m s . The d iscrepancy between the saponif icat ion

equivalent and the acetyl content remains obscu re . Es t i

mation of the sucrose content by the anthrone method

- 5 7 -

gave v a l u e s of 8 9 . 6 and 9 0 . 0 p e r c e n t s u c r o s e ( t h e o r e t i c a l

for s u c r o s e m o n o a c e t a t e i s 8 9 . 2 p e r c e n t ) . R e p r e c i p i t a t i o n

f rom dry n - b u t a n o l did not change the m a t e r i a l a p p r e c i a b l y

C h r o m a t o g r a p h y on p a p e r u s ing the n - b u t a n o l / w a t e r s y s t e m

showed the p r e s e n c e of four wel l def ined s p o t s ; Rf v a l u e s :

0 . 0 5 , 0 . 1 0 , 0 .146 and 0 . 2 8 3 . On the s a m e p a p e r , s u c r o s e

had an Rf v a l u e of 0 . 0 5 . An i l ine p h o s p h a t e was used as

s p r a y r e a g e n t .

The m a t e r i a l was s u b j e c t e d to e x t r u s i o n p a r t i t i o n

c h r o m a t o g r a p h y on C e l i t e u s i n g n - b u t a n o l / w a t e r as d e v e l o ­

p ing s o l v e n t . The co lumn was s p r a y e d with a l k a l i n e p e r ­

m a n g a n a t e (75) to r e v e a l only t h r e e bands which w e r e e lu ­

t ed wi th e t h a n o l . The r e c o v e r y was 67 p e r c e n t . The

w e i g h t s of t he f r a c t i o n s and t h e i r a c e t y l c o n t e n t s a r e l i s ­

t ed in T a b l e IV .

T a b l e IV

S e p a r a t i o n of a M i x t u r e of S u c r o s e A c e t a t e s on C e l i t e .

F r a c t i o n Rf Value Weight Acety l Conten t Acety l Groups in m g . in m . e . p e r mo le

1

2

3

T o t a l

0 .05 (a) 3 2 . 0

. 35 .4 [ 0 . 1 0 (b) i; (0 146

0 . 2 8 3 ( c ) 26.7

194. 1 ( d )

0. 024

0 .298

0 110

0 .432

0 .26

0 .83

1 .71

0 .84

-58 -

( a ) . The fract ion was sucrose with a t r a ce of ma te r i a l with

Rf value about half of that of s u c r o s e .

( b ) . The m a t e r i a l was contaminated with a small amount of

sucros e.

( c ) . The m a t e r i a l was contaminated with a small amount of

f ract ion 2 together with a very small amount of a subs­

tance with a g r e a t e r R* va lue , presumably sucrose t r i ­

ace ta te .

( d ) . Sixty g. of Celi te were used to chromatograph 280 mg.

of the suc rose ace ta te in a column 36 mm. in d iameter

These data show that the sucrose ace ta te studied

was a mix ture cons is t ing of approximately 17 percen t su­

c r o s e , 69 percent suc rose monoaceta te , 14 percent sucro­

se d iace ta te and some sucrose t r i a c e t a t e . The fact that

the sample analyzed as a monoacetate was en t i re ly for­

t u i t o u s . The Celi te chromatogram together with the r e ­

sul ts of the paper chr omatograms show c lea r ly that the

main component (approximate ly 69 percen t ) was a mixture

of i somer ic sucrose monoaceta tes .

- 5 9 -

I I I . DISCUSSION OF EXPERIMENTAL RESULTS.

At the beg inn ing of t h i s i n v e s t i g a t i o n in e a r l y May

1955, no r e f e r e n c e could be found in the c h e m i c a l l i t e r a t u ­

r e r e l a t i n g to the e s t e r i f i c a t i o n of s u c r o s e by t r a n s e s t e r i ­

f i c a t i o n . P r e l i m i n a r y s t u d i e s had been m a d e , h o w e v e r , by

F . D . Snel l and c o w o r k e r s .

The p r o c e d u r e for the a c y l a t i o n of s u c r o s e by

t r a n s e s t e r i f i c a t i o n employed t h roughou t t h i s work ( s e e p .

31) was b a s e d on a m e m o r a n d u m sen t to us by L . Osipow

(81) of the Snel l g r o u p . Th i s m e t h o d , which employed

a e r a t i o n to r e m o v e the m e t h a n o l b y p r o d u c t , was modi f ied

in the l a b o r a t o r y to exc lude the p a s s a g e of a i r . The

h i g h e r vacuum used in the p r o c e d u r e employed in t h i s work

was a d e q u a t e to r e m o v e the m e t h a n o l . P o t a s s i u m c a r b o n a ­

te had been found by the Snel l group to be the b e s t c a t a l y s t

for t h i s p a r t i c u l a r r e a c t i o n . It was employed in t h i s i n ­

v e s t i g a t i o n to the e x c l u s i o n of o t h e r s . The DMF so lven t

was chosen for s i m i l a r r e a s o n s .

The method of i s o l a t i o n of the c r u d e m y r i s t a t e

e s t e r p r o d u c t , u s ing n - b u t a n o l or m e t h y l e thyl ke tone and

f ive p e r c e n t aqueous sod ium c h l o r i d e to p a r t i t i o n the r e ­

a c t i o n c a k e , was deve loped in t h i s l a b o r a t o r y . It was l a ­

t e r adop ted by o the r w o r k e r s ( 4 1 ) .

-60-

Prepara t ions of sucrose s t e a r a t e s , pa lmi ta tes and

m y r i s t a t e s done in the ear ly s tages of this work have r e ­

sulted in the l a t t e r being adopted exclusively for the r e ­

mainder of the s tudy. This choice was based on the fact

that the sucrose m y r i s t a t e s possessed a super io r hydrophi-

le- lypophi le balance and consequent ly , the p r o p e r t i e s of

the const i tuents ( suc rose and m y r i s t i c acid) are well mas ­

ked.

1 . Prepara t ions of Sucrose Myr i s ta tes by T r a n s e s t e r i f i c a ­

tion .

The r e s u l t s of a number of p repa ra t ions of suc ro ­

se myr i s t a t e s using the procedure given on page 31 are

l i s ted in Table V. The pur i f ica t ion procedures for the

samples l i s ted a re given in the footnotes to Table V.

The r e s u l t s p resen ted in Table V confirm a v a r i e ­

ty of observat ions r epo r t ed by Snell and coworkers (41) .

It is to be noted f i r s t of a l l , that the crude products from

the shor t e r reac t ion t imes have the higher e s t e r group

con ten t s . The products i so la ted after eight hours or more

of reac t ion time all possessed approximately 1.1 m y r i s ­

toyl groups per suc rose r e s i d u e . The acylat ion of s u c r o ­

se by t r a n s e s t e r i f i c a t i o n with methyl myr i s t a t e the re fore

definitely goes through a d i e s t e r concentra t ion maximum

6 1 -

T a b l e V

P r e p a r a t i o n s of S u c r o s e M y r i s t a t e s by T r a n s e s t e r i f i c a t i o n

Sample (a)

1

2

2a

3

3a

4

5

5a

5b

6

6a

Methyl myristate

mM(b)

50

175

175

175

175

5 8 . 3

Reaction time in hours

13

8

1 1

7

6 . 5

1 1 . 5

Saponi­fication

equiv.

515

521

520

520

552

478

408

350

528

512

507

Fatty acid equiv.

-

-

528

528

-

480

414

-

534

519

513

Myristoyl groups per molecule

-

-

1.1

1 .1

1.0

1.27

1.72

2 .44

1 .08

-

1 . 13

Percent yield

(c)

88

85

-

72

-

60

74

-

-

90

-

( a ) . The s a m p l e s w e r e i s o l a t e d and p u r i f i e d as f o l l o w s .

1 . I s o l a t e d u s i n g me thy l e thy l k e t o n e .

2 . I s o l a t e d u s i n g me thy l e thy l k e t o n e .

2 a . Sample 2 (76 g . ) was d i s s o l v e d in 220 m l . of DMF-wa

t e r (10 :1) and e x t r a c t e d c o n t i n u o u s l y wi th hexane for

24 h o u r s . E v a p o r a t i o n of the DMF p h a s e y i e l d e d t h i s

p r o d u c t . Pape r c h r o m a t o g r ams deve loped with n - b u -

t a n o l / w a t e r and s p r a y e d with a n i l i n e p h o s p h a t e s h o -

- 6 2 -

F o o t n o t e s to T a b l e V.

wed t r a c e s of s u c r o s e to be p r e s e n t .

I s o l a t e d u s i n g m e t h y l e thy l ke tone and p u r i f i e d as des

c r i b e d above for s a m p l e 2 a .

a . Sample 2a and 3 w e r e combined (90 g . ) , d i s s o l v e d in

350 m l . of n - b u t a n o l and e x t r a c t e d with f ive p e r c e n t

aqueous sodium c h l o r i d e to r e m o v e the t r a c e s of s u ­

c r o s e . The bu tano l p h a s e y i e l d e d a so l id which was

p r e c i p i t a t e d twice from 400 m l . of a c e t o n e to g ive

57 g . of p r o d u c t , (0Q9 + 4 2 . 6 ( c . 1 in m e t h a n o l ) , sof-

* 9

t e n i n g p o i n t , 65 -70 C ) . The a m o r p h o u s so l i d was

ob ta ined as m i c r o s p h e r i c a l g r a n u l e s . T h i s e s t e r con ­

t a i n e d 5 7 . 5 p e r c e n t s u c r o s e as d e t e r m i n e d with an ­

t h r o n e ( v a l u e for m o n o e s t e r is 6 2 . 0 p e r c e n t s u c r o s e ) .

V o l a t i l e a m i n e , 28 m . e . , was d e t e c t e d in the t r a p .

The 7 3 . 6 g . of c r u d e e s t e r i s o l a t e d u s i n g n - b u t a n o l ,

was d i s s o l v e d in 350 m l . of DMF. When t h i s s o l u t i o n

was s a t u r a t e d with h e x a n e , 1.5 g . of p o t a s s i u m soap

p r e c i p i t a t e d . After f i l t r a t i o n , 35 m l . of w a t e r w e r e

added to the DMF and the s o l u t i o n was e x t r a c t e d con­

t i n u o u s l y with hexane for 21 h o u r s . T h i s r e m o v e d

9 .75 g . of h igh ly s u b s t i t u t e d s u c r o s e m y r i s t a t e s ( f a t ­

ty ac id e q u i v a l e n t of 357 g . ) . E v a p o r a t i o n of the DMF

gave 62 g . of p r o d u c t which was p r e c i p i t a t e d from

- 6 3 -

F o o t n o t e s to T a b l e V.

a c e t o n e to g ive 50 g . of t h i s p r o d u c t .

5 . V o l a t i l e a m i n e , 23 m . e . was d e t e c t e d in the t r a p . Be­

f o r e r e m o v i n g the DMF, the r e a c t i o n m i x t u r e was ex ­

t r a c t e d f ive t i m e s with 200 m l . of h e x a n e - T h i s t r e a t ­

ment r e m o v e d 1.3 g . of s o l i d . The 6 6 . 1 g . of c r u d e

e s t e r p r o d u c t i s o l a t e d u s i n g me thy l e thyl k e t o n e , was

p r e c i p i t a t e d f rom 300 m l . of a c e t o n e to g ive 53 g . of

t h i s p r o d u c t .

5 a . Sample 5 (25 g . ) was p r e c i p i t a t e d f rom 125 m l . of

m e t h a n o l to y i e l d 1 2 . 7 1 g . of t h i s m a t e r i a l .

5b . The m e t h a n o l m o t h e r l i q u o r s from the p r e p a r a t i o n of

s a m p l e 5a y i e lded 11 .27 g . of so l id which was p r e c i ­

p i t a t e d f rom 50 m l . of a c e t o n e to y i e l d t h i s m a t e r i a l .

6 . I s o l a t e d u s ing n - b u t a n o l .

6 a . A f ive t r a n s f e r c o u n t e r - c u r r e n t d i s t r i b u t i o n was done

on 10 g . of s a m p l e 6 with the so lven t s y s t e m DMF:

w a t e r : d i e thy l e t h e r ( 1 : 1 : 2 ) . T h r e e t ubes w e r e u s e d

and 100 m l . of each p h a s e was employed p e r t u b e .

The combined bo t tom p h a s e s of t ubes 0 , 1 and 2 gave

o upon e v a p o r a t i o n in vacuo at 50 C . , 7 .5 g . of p r o d u c t

which a f forded 6 .17 g . of whi te so l id a f t e r p r e c i p i t a ­

t ion from 45 m l . of a c e t o n e . Pape r c h r o m a t o g r a m s

showed tha t t r a c e s of s u c r o s e w e r e p r e s e n t . T h e s e

- 6 4 -

Footnotes to Table V.

t r a c e s were removed by ex t rac t ing a n-butanol solu­

tion of the e s t e r with five percent sa l t so lu t ion . Eva­

porat ion of the butanol gave this s ample .

(b ) . In each p r e p a r a t i o n , there was a two molar excess

of s u c r o s e . The potass ium carbonate (0 .1 molar l e ­

vel based on the methyl myr i s t a t e ) and the DMF ( 3 . 3

m l . per g. of sucrose) were used in the same r e l a t i ­

ve amounts throughout .

( c ) . The yields are based on the methyl myr i s t a t e and ca l ­

culated using the analyses of the products by saponi­

fication .

during the f i r s t seven hours of the reac t ion with thermody­

namic equi l ibr ium being reached only after about eight

hours of reac t ion t i m e . Such a course for the e s t e r i f i c a ­

tion can only be due to the in i t ia l ly formed monoes te rs

undergoing es te r i f i ca t ion at a much g r e a t e r r a t e than su­

c r o s e .

The p repa ra t ion of samples 4 and 5 show that ap­

p rec iab le amounts of dimethylamine a re formed. This

would presuppose side r eac t ions occur r ing with the solvent

In view of the need of the sucrose e s t e r de te rgen t s in the

pharmaceu t i ca l and food i n d u s t r i e s , the p r e sence of the

-65 -

toxic formate group in these products would have been a

se r ious objection to the manufacturing p rocess employing

DMF as solvent . Such formate e s t e r s could r e su l t from

the reac t ion of sucrose with DMF according to the follo­

wing equat ion.

K2C03 R-OH -H HCO-N(CH3)a ^ > HCO-OR +• HN(CHj)z

Although the agreement between the sapinif icat ion equiva­

lents and the fatty acid equivalents of the e s t e r s e l imina­

ted the poss ib i l i ty of significant levels of the formyl group,

one must remember that an analyt ica l method is only as

good as what is looked for .

The r e s u l t s of the de terminat ions for formyl group

c a r r i e d out on the sucrose m y r i s t a t e s employing the ana­

ly t ica l method developed during this study (p . 35), have

defini tely shown that , under the conditions of the t r a n s e s ­

te r i f i ca t ion descr ibed on page 31 , reac t ion t imes of 15

hours gave sucrose myr i s t a t e samples containing less than

0.1 percent by weight of formyl group. This can be seen

from the fact that 913.4 mi l l ig rams of sucrose myr i s t a t e

containing 1.24 myr is toyl groups per sucrose molecule

contained 0.0216 mi l l imoles of formyl r e s i d u e . Other s i ­

de reac t ions possibly undergone by the solvent were not

inves t iga ted .

- 6 6 -

The r epo r t s of Snell (41) concerning the p resence

of potassium soap in the sucrose fatty e s t e r s were conf i r ­

med during the p resen t work. The 1.5 grams of soap i so ­

lated in the p repa ra t ion of sample 4 r e p r e s e n t s 3.2 pe r ­

cent of the methyl myr i s t a t e or ig inal ly used . Other de t e r ­

minat ions have shown that after 15 hours of reac t ion t ime ,

4.7 percent of the methyl e s t e r had been converted to soap .

The potass ium soap i so la ted was analyzed by doing fatty

acid contents and weighing the combustion r e s i d u e s . It

was not determined whether this soap was formed during

the es te r i f i ca t ion or during the i so l a t ion .

It was f i r s t at tempted to sepa ra t e the po lyes t e r s

from the monoes ters by ext rac t ion of the reac t ion mixture

with hexane . The p repara t ion of sample 5 showed this to

be not ef fect ive . The p repara t ion of sample 4 shows that

the ex t rac t ion of a DMF-water (10:1) solution of the crude

e s t e r product can be used to remove the t r i - and higher

e s t e r s .

A solution to the problem of separa t ing mono-

from d i e s t e r s was provided by c o u n t e r - c u r r e n t d i s t r i b u ­

t ion . P re l iminary s tudies with the solvent system DMF:

water :d ie thy l ether (1:1:2) had shown that the di - and

higher sucrose m y r i s t a t e s were removed rapidly by the

- 6 7 -

success ive ether ex t rac t ions while the monomyr is ta tes

remained in the bottom layers of the f i r s t th ree tubes .

Thus , a coun t e r - cu r r en t d i s t r ibu t ion of one gram of sam

pie 5 (or ig ina l ly containing 1.72 myr is toy l groups per

molecule of sucrose) using 50 ml . of each phase gave a

po lyes te r f ract ion which contained 2.76 groups per sucrose

r e s i d u e . Evaporat ion of the f i r s t th ree bottom l aye r s

gave a solid which appeared to possess 1.05 acyl groups

when analyzed by saponificat ion but had a fatty acid equi­

valent indicat ing only 0.91 g roups . The d iscrepancy bet ­

ween the saponification and the fatty acid equivalents ex­

per ienced with the products i sola ted from the bottom l aye r s

of these d i s t r ibu t ions is probably re la ted to the es te r

molecules undergoing chemical change during the e x t r a c ­

tion and isola t ion p r o c e s s e s . Paper chromatography showed

that sucrose was l ibera ted during these d i s t r i b u t i o n s .

Some hydro lys i s of the e s t e r s therefore occu r r ed . The r e ­

sul ts obtained in the p repa ra t ion of sample 6 show that

pure sucrose e s t e r s were obtained when these f ract ions

were p rec ip i t a t ed from ace tone .

Two solvents were found to be useful for the pu­

r i f ica t ion of sucrose m y r i s t a t e s by p rec ip i t a t ion p r o c e ­

d u r e s . The p repa ra t ion of sample 5a shows that p r e c i p i ­

ta t ion from methanol will remove the m o n o e s t e r s . On the

- 6 8 -

o t h e r hand , p r e c i p i t a t i o n from a c e t o n e as in the p r e p a r a ­

t ion of s a m p l e 3a r e s u l t e d in an e n r i c h m e n t in s u c r o s e

m o n o m y r i s t a t e s . T h e s e a c e t o n e p u r i f i c a t i o n s w e r e c a r ­

r i e d out by d i s s o l v i n g the e s t e r in b o i l i n g a c e t o n e , f i l t e ­

r i n g the hot s o l u t i o n and coo l ing the f i l t r a t e to b r i n g about

p r e c i p i t a t i o n of the m o n o e s t e r s . Tha t the p r o c e d u r e was

a c c o m p a n i e d by a s m a l l amount of c o n v e r s i o n of s u c r o s e

m o n o e s t e r s in to d i e s t e r s and s u c r o s e was i n d i c a t e d by the

p r e c i p i t a t i o n of s u c r o s e whi le d i s s o l v i n g the s u c r o s e f r e e

e s t e r s in the hot a c e t o n e . T h i s c o n v e r s i o n did not a lways

o c c u r and p e r h a p s i s r e l a t e d to t r a c e s of a l k a l i n i t y .

2 . P r e p a r a t i o n s of S u c r o s e M y r i s t a t e s Using the Acid

C h l o r i d e .

Two p r e p a r a t i o n s of s u c r o s e m y r i s t a t e s w e r e

made u s i n g the p r o c e d u r e d e s c r i b e d on page 3 3 . The r e

s u i t s a r e p r e s e n t e d in T a b l e VI .

The r e s u l t s g iven in T a b l e VI show c l e a r l y t h a t ,

as in the p r e p a r a t i o n of s u c r o s e m y r i s t a t e s by t r a n s e s t e ­

r i f i c a t i o n , the s u c r o s e m o n o m y r i s t a t e s fo rmed by the

ac t i on of m y r i s t o y l c h l o r i d e in p y r i d i n e a r e e s t e r i f i e d m o ­

r e r a p i d l y than is s u c r o s e . S i m i l a r r e s u l t s have been ob­

s e r v e d when s u c r o s e was a c e t y l a t e d with a c e t i c a n h y d r i d e

in p y r i d i n e ( 8 5 ) . The a c y l a t i o n in p y r i d i n e is i r r e v e r s i b l e

69

T a b l e VI

P r e p a r a t i o n s of S u c r o s e M y r i s t a t e s u s i n g t h e A c i d C h l o r i d e

Sample (a)

7a

7b

8

8a

8b

Sucrose mM

73 .2

1 0 9 . 8

Myristoyl chloride

mM

3 6 . 6

3 7 . 6

Saponi­fication equiv.

384

311

539

365

320

558

Fatty acid

equiv.

391

365

333

534

Myristoyl groups per

molecule

1 . 9 6

3 . 3 9

1 . 0 4

2 . 2 1

2 . 7 7

1 . 0 5

Percent yield

(b)

93

92

( a ) . T h e s a m p l e s w e r e i s o l a t e d a n d p u r i f i e d a s f o l l o w s .

7 . I s o l a t e d a s d e s c r i b e d on p a g e 3 3 , t h e s u b s t a n c e g a v e

a n e g a t i v e F e h l i n g ' s t e s t .

7 a . S a m p l e 7 (10 g . ) w a s p r e c i p i t a t e d f r o m 65 m l . of a

m i x t u r e of e t h a n o l and m e t h a n o l ( 1 : 5 ) . T h e y i e l d w a s

4 . 3 5 g . T h e m a t e r i a l c o n t a i n e d no f r e e a c i d and w a s

s o l u b l e in h e x a n e .

7 b . T h e m o t h e r l i q u o r s f r o m t h e p r e p a r a t i o n of s a m p l e

7a y i e l d e d 4 . 8 9 g . of s o l i d w h i c h w a s p r e c i p i t a t e d

f r o m 70 m l . of a c e t o n e . T h e y i e l d w a s 2 . 7 9 g .

8 . T h e m a t e r i a l , i s o l a t e d a s d e s c r i b e d on p a g e 3 3 , w a s

f r e e of s u c r o s e and g a v e a n e g a t i v e F e h l i n g ' s t e s t .

8 a . A f i v e t r a n s f e r c o u n t e r - c u r r e n t d i s t r i b u t i o n w a s m a d e

- 7 0 -

F o o t n o t e s to T a b l e VI .

wi th 10 g . of s a m p l e 8. The so lven t s y s t e m was

D M F : w a t e r : d i e t h y l e t h e r ( 1 : 1 : 2 ) . T h r e e t ubes w e r e

u sed and 200 m l . of each p h a s e was employed p e r

t u b e . The e x t r a c t i o n s had to be c e n t r i f u g e d to b r e a k

the e m u l s i o n s . The combined top p h a s e s of t ubes 5 ,

4 and 3 gave 7 .0 g . of t h i s s u c r o s e f r e e m a t e r i a l .

8 b . C o n c e n t r a t i o n of the bo t tom p h a s e s of t ubes 0, 1 and

2 f rom above gave 3 .0 g . of a da rk r e s i d u e shown by

p a p e r c h r o m a t o g r a p h y to con ta in s u c r o s e . T h i s s o ­

l id was t r e a t e d with 20 m l . of b o i l i n g a c e t o n e , f i l ­

t e r e d and a 1.56 g . c rop of whi te so l id was ob ta ined

on coo l ing the f i l t r a t e . Th i s so l id was d i s s o l v e d in

n - b u t a n o l and e x t r a c t e d with f ive p e r c e n t s a l t s o l u ­

t ion to r e m o v e the t r a c e s of s u c r o s e s t i l l p r e s e n t .

The m a t e r i a l was r e c o v e r e d by e v a p o r a t i o n of the

b u t a n o l .

( b ) . The y i e l d s a r e b a s e d on the m y r i s t o y l c h l o r i d e and

w e r e c a l c u l a t e d u s ing the m y r i s t o y l con ten t d e t e r ­

mined by s a p o n i f i c a t i o n .

and , in c o n t r a s t wi th the t r a n s e s t e r i f i c a t i o n , the f i r s t

p r o d u c t s of t he r e a c t i o n a r e t h o s e i s o l a t e d .

I t i s no t ewor thy tha t the p u r i f i c a t i o n of s a m p l e

- 7 1 -

8b r e s u l t e d in e x t e n s i v e d a r k e n i n g and h y d r o l y s i s . Th i s

s a m p l e , which a n a l y z e d at 1.05 m y r i s t o y l g roups p e r m o ­

l e c u l e of s u c r o s e is t h e r e f o r e p r o b a b l y not r e p r e s e n t a t i v e

of the o r i g i n a l m o n o m y r i s t a t e s a m p l e .

3 . The Rate of the T r a n s e s t e r i f i c a t i o n R e a c t i o n .

Snel l and c o w o r k e r s (41) have done rough r e a c t i o n

k i n e t i c s of the p r e p a r a t i o n of fa t ty e s t e r s of s u c r o s e by

t r a n s e s t e r i f i c a t i o n . They r e p o r t e d t h a t , unde r r e a c t i o n

c o n d i t i o n s which c o m p a r e c l o s e l y to t h o s e employed du­

r i n g t h i s work ( s e e p . 15) , the me thy l fa t ty e s t e r had com­

p l e t e l y r e a c t e d a f t e r four to s ix h o u r s of r e a c t i o n t i m e .

The a l i q u o t s w i thd rawn from the r e a c t i o n m i x t u r e s w e r e

d r i e d u n d e r r e d u c e d p r e s s u r e at 100 C. b e f o r e be ing a n a ­

l y z e d . T h i s i s o l a t i o n s tep undoub ted ly changed the compo­

s i t i o n of the s a m p l e w i thd rawn from the r e a c t i o n m i x t u r e .

I t was t h e r e f o r e dec ided to t r a n s f e r a l i q u o t s of the r e a c ­

t ion m i x t u r e d i r e c t l y to co lumns for c h r o m a t o g r a p h i c a n a ­

l y s i s . The r e s u l t s p l o t t e d in F i g . 2 i n d i c a t e tha t the t r a n s ­

e s t e r i f i c a t i o n of me thy l m y r i s t a t e with s u c r o s e was com­

p l e t e in seven to e igh t h o u r s of r e a c t i o n t i m e . T h i s r e s u l t

i s in a g r e e m e n t wi th the r a t e s s u g g e s t e d by the da ta r e p o r ­

ted in T a b l e V. I t was not p o s s i b l e to con t inue s t u d i e s

a long t h i s l i n e and the r e a s o n for the low r e c o v e r i e s of the

- 7 2 -

aucrose remains obscu re . It is felt that this approach for

the study of the k inet ics of the t r a n s e s t e r i f i c a t i o n reac t ion

war r an t s fur ther a t t en t ion .

4. The S t ruc tu re of the Sucrose E s t e r s .

In view of our inabi l i ty to separa te the sucrose

monomyr i s ta tes into the posi t ional i s o m e r s , these com

pounds were examined as m i x t u r e s .

a. Determinat ion of the Number of Myris toyl

Groups Occupying the 6- and 6 ' - Posit ions in a Sucrose

M y r i s t a t e . - The r e s u l t s obtained on t rea tment of the to ­

syl e s t e r s of sucrose and sucrose der iva t ives with sodium

iodide in acetone a re l i s ted in Table VII. The compounds

tosy la ted a re those r epor t ed , using the same numbers , in

Tables V and VI. F igure 3 is a plot of the iodine atoms

introduced into a tosylated sucrose monomyris ta te (Table

VII, compound 3a) after a var ie ty of reac t ion t i m e s . Si­

mi la r r e s u l t s obtained with tosylated sucrose (Table VII)

a re included.

A kinet ic study of the iodination reac t ion (F ig . 3)

showed that all the rep laceab le tosyloxy groups had r e a c ­

ted after eight h o u r s . Reaction t imes longer than 15

hours r e su l t ed in secondary reac t ions taking place as in-

T a b l e V I I

T o s y l a t i o n a n d I o d i n a t i o n E x p e r i m e n t s

Compound Myristoyl groups per molecule

T o s y l a t i o n I o d i n a t i o n Percent Percent Groups Reaction Percent Percent sulfur yield introduced t ime, h r s . iodine yield

(a) (a)

Myristoyl Atoms groups at

introduced 6 - a n d 6 ' -

Sucrose 14.93 91 5.67 19.55 85 1.75

13 22.55 94 2.00

Methyl -D-gluco-pyranoside (b) 14.29 98 2.76

13

25.62 72

28.28

1.15

1 .25

i

CO i

2 , 3 , 6 , 3 ' , 4 ' -penta-O-acetyl sucrose (c) 9.35 2.93 13.40 97 1.02

Sucrose myr i s t a t e s : 3a 1.00 12.33 84 5.24 2

4

8

13

11.72

14.11

14.82

15.03

94

87

-

89

1.21

1.44

1.51

1.53 0.47

4 1.27 - 11.23 88 4.65 8 12 .91 - 1.29 0 .71

Table VII (cont inued) .

Compound

Sucrose myristJ 5a

5b

6a

7a

7b

8a

8b

Myristoyl groups per molecule

ites: 2.44

1.08

1.13

3.39

1.04

2.77

1.05

T Percent sulfur

8.66

11 .90

12.00

5.95

11.45

6.91

11.92

o s y l a t i o n Percent

yield (a)

86

80

85

80

65

68

L

Groups introduced

3.96

4.93

5.14

2.74

4.45

2.98

4.91

Reaction time, h r s .

8

8

8

8

7

8

8

I o d i n a t i o n Percent

iodine

8.35

15 .22

14.48

4.99

13.86

5.70

16.89

Percent yield (a)

93

97

93

82

89

73

80

Atoms ' introduced

0.94

1.52

1.49

0.57

1.30

0.61

1.66

M

6-

yristoyl •oups at - and 6*-

1.06

0.48

0.51

1.43

0.70

1.39

0.44

( a ) . The yie lds were based on the theo re t i ca l amounts expected from the a n a l y s e s . o r -.13 o

(b ) . Melting point , 165.5 C . , r otat ion, \<\ J_ +158 ( c . 8 in water )

( c ) . Kindly provided by Mr. J . P . Bar re t t e

- 7 5 -

2 . 0 . . O

( 1 )

<u

o 2 u 0) P.

• c a> o

•a o u

1.6--

1.2..

( 2 )

CO

6 o

9i a

O 0 . 8 - .

0 . 4 . . ( 1 ) . S u c r o s e 5 . 6 7 - T o s y l a t e .

( 2 ) . S u c r o s e M o n o m y r i s t a t e 5 . 2 4 - T o s y l a t e .

+ + + + 6 8

T i m e in Hour s 10 12

F i g . 3 . The I o d i n a t i o n of the T o s y l a t e s of S u c r o s e and of S u c r o s e M o n o m y r i s t a t e .

- 7 6 -

d i c a t e d by the l i b e r a t i o n of m o r e and m o r e i o d i n e . The

i o d i n a t i o n s w e r e t h e r e f o r e s topped a f t e r e ight h o u r s .

It is wel l e s t a b l i s h e d ( 5 6 , 57) tha t p r i m a r y t o s y l ­

oxy g r o u p s a r e u s u a l l y r e a d i l y r e p l a c e d by iod ine when

the compound is t r e a t e d with sodium iod ide in a c e t o n e .

T h u s , 1.25 tosy loxy g r o u p s of a methy l OC-D-glucopyr ano -

s i d e 2 . 7 6 - t o s y l a t e u n d e r w e n t r e p l a c e m e n t . Th i s high r e ­

s u l t was p r o b a b l y due to e l i m i n a t i o n of s e c o n d a r y t o sy l

g r o u p s in the c o u r s e of the r e a c t i o n . C e r t a i n l y , the r e ­

s u l t s i n d i c a t e tha t one of the t o sy loxy g roups was m o r e

r e a d i l y r e p l a c e d than the o t h e r s . I od ina t i on of the s u c r o ­

se 5 . 6 7 - t o s y l a t e led to the r e p l a c e m e n t of two of the t o s y l ­

oxy g r o u p s . Th i s fac t t o g e t h e r wi th the fac t t ha t 4 , 1 ' , 6 ' -

t r i - O - t o s y l s u c r o s e p e n t a a c e t a t e unde rwen t r e p l a c e m e n t

of only one of the t o sy loxy g r o u p s shows tha t the 6 - t o s y l -

oxy g roup of a s u c r o s e t o s y l a t e i s r e a d i l y r e p l a c e d by i o d i ­

n e . A l s o , the r e s u l t s show tha t e i t h e r the 1 ' - or 6 ' - t o s y l -

oxy g roup of a s u c r o s e t o s y l a t e i s r e p l a c e a b l e by i o d i n e .

Tha t the r e p l a c e a b l e t o s y l o x y group i s tha t s i t u a t e d at the

6 ' - p o s i t i o n i s c l e a r l y i n f e r r e d by the s t e r i c r e q u i r e m e n t s

for the i o d i n a t i o n r e a c t i o n . It i s wel l e s t a b l i s h e d t ha t t h e ­

se r e a c t i o n s a r e of the S^ 2 type and p r o c e e d by way of an

a t t a c k at the r e a r s i d e of the c a r b o n c a r r y i n g the t o s y l o x y

g r o u p . Ingold and c o w o r k e r s (83) have shown tha t such an

- 7 7 -

at tack is s t rongly prohibi ted in the case of neopentyl h a l i -

d e s . The s i tuat ion at the 1' posi t ion of a sucrose tosy la ­

te c losely r e sembles that in a neopentyl hal ide and, conse­

quently can be expected to be inact ive for s imi l a r r e a s o n s .

That this is in fact the case is supported by the observat ion

that 2, 3; 4, 5-di -O - is opropylidene-/?-D-fructose tosyla te is

s table to iodination (84) .

On the bas is that only the 6- and 6 ' - tosyloxy

groups in a sucrose tosyla te are rep laceab le by iodine, the

iodinat ions of the tosy la tes of samples 3a, 5b, and 6a e s ­

tabl i sh that approximately half of the acyl groups of these

suc rose monomyris ta tes a re at the 6- and 6 ' - pos i t i ons .

R e - i n t e r p r e t a t i o n of the r e su l t s obtained by Snell and co­

workers (61) (p . 27) indica tes that only 37 percent of the

acyl groups of the sucrose monolaurate studied were at the

6-and 6—posit ions.

The tosylat ion and iodination exper iments pe r fo r ­

med on the sucrose monomyris ta te obtained by acylation

with myr i s toy l chlor ide (sample 7b, Table VII) suggest

that 70 percent of the myr is toy l groups were at the 6 and

6 ' - p o s i t i o n s . Thus , on the bas is of this exper iment , it

appears that the monoester p repa red by acylat ion p o s s e s ­

ses approximately 0.2 more myr is toy l groups at the 6- and

- 7 8 -

6 ' - p o s i t i o n s . This is reasonable since these p r imary po­

s i t ions can be expected to undergo acylat ion more rapidly

than the other more hindered pos i t i ons . However, the r e ­

sul ts with sample 8b (Table VII) throw some doubt on this

conclus ion. It is to be noted, however, that the p r e p a r a ­

tion of sample 8b was accompanied by extensive hydrolys is

in the course of the counter - cu r ren t d i s t r i bu t ion . Thus

it may prove that myr i s toy l groups at the 6- and 6 ' - pos i ­

t ions are more suscept ib le to hydrolys is than those at the

other pos i t ions .

b . Per iodate Ox ida t ions . - The per iodate oxidation

of a suc rose der iva t ive can be used to obtain information

regard ing the na ture of the posi t ional i somer or i somers

p resen t in the d e r i v a t i v e . This is seen from Table VIII

which gives the theore t i ca l values for the consumption of

pe r ioda te and the production of formic acid when the va­

r i e ty of poss ib le mono-O-subs t i tu ted sucroses are oxidized

by the r eagen t .

The oxidation of 72.88 mi l l ig rams of pure s u c r o ­

se monomyris ta te p repared by t r anse s t e r i f i c a t i on (sample

3a, Table V) r e su l t ed in 2.87 mi l l imoles of per ioda te

being consumed after two days . The r e s u l t s of th is oxida­

tion a re given in Table IX.

7 9 -

T a b l e VIII

The Consumpt ion of P e r i o d a t e and the P roduc t i on of F o r m i c

Acid for the P o s s i b l e Mono-O - S u b s t i t u t e d S u c r o s e s .

P o s i t i o n of S u b s t i t u e n t

g roup

6, 1' or 6'

2 or 4

3 ' or 4*

Mola r e q u i v a l e n t of p e r i o d a t e consumed

Mola r equ iva l en t of f o r m i c acid

p r o d u c e d

0

0

1

T a b l e IX o

P e r i o d a t e Ox ida t ion of S u c r o s e M o n o m y r i s t a t e at 2 4 . 8 C

R e a c t i o n h o u r s

0 .

0 .

3

4

24

4 8

3

5

t i m e , •

mM of sodium p e r i o d a t e p e r mM of s u c r o s e m y r i s t a t e .

0 .36

0 .89

1 .43

1.66

2 .08

2 .87

The r a t e of up t ake of oxidant was s l o w e r for the

s u c r o s e e s t e r s than for s u c r o s e b e c a u s e p a r t of the e s t e r

- 8 0 -

was d i spersed as a fine col loidal suspension in the oxida­

tion media . Extensive hydro lys i s of the es te r l inkage was

ru led out, s ince after th ree days , the reac t ion mixture

s t i l l had the p rope r t i e s of a detergent solution and the

fine amorphous p rec ip i t a t e which had formed could be r e a ­

dily dissolved in aqueous methanol with foaming. Apparent­

ly, pa r t of the oxidation product had p rec ip i t a ted out of

so lu t ion .

Attempts to oxidize sucrose monomyris ta te sam­

ples in 50 percent aqueous methanol , in order to i nc r ea se

the solubi l i ty of the e s t e r s in the reac t ion media, have

r e su l t ed in consumptions of per iodate exceeding three

mi l l imoles per mi l l imole of e s t e r . Thus , sample 5b (Ta

ble V), which contained 1.08 myr is toyl groups per molecu­

l e , consumed 3.19 mi l l imoles of oxidant after two days .

This r e s u l t , which must be due to pa r t i a l hydrolys is of

the glycosidic l inkages , cas t s some uncer ta in ty on the

value of the method for studying the s t r u c t u r e of sucrose

e s t e r s . N e v e r t h e l e s s , the r e s u l t s support the conclusion

reached by Snell and coworkers (61) that the monoes te r s

obtained by t r a n s e s t e r i f i c a t i o n are subst i tu ted almost en­

t i r e l y at the p r imary posi t ions of s u c r o s e . This s t ructu

re for a suc rose monoester p repared by t r a n s e s t e r i f i c a t i o n

would not be s u r p r i s i n g since it is well es tab l i shed (86)

- 8 1 -

that acyl groups have a s t rong tendency to migra te to p r i ­

mary posi t ions especia l ly in alkal ine media as is used in

the t r a n s e s t e r i f i c a t i o n .

In view of the above r e s u l t s , it can be concluded

with a ce r ta in degree of ce r t a in ty that the myris toyl

groups of the suc rose monomyris ta te are subs tant ia l ly en­

t i r e l y s i tuated at the th ree p r imary pos i t i ons . Since ap­

proximate ly one-half of the groups were shown to be at

the 6- and 6' pos i t ions by the tosylat ion and iodination

method of ana ly s i s , it follows that the other half of the

groups are at the 1 ' - pos i t ion .

c . The Reduction of Tosyl E s t e r s . - K a r r e r and

coworkers (79, 87) have shown that in some c a s e s , the

l i thium aluminum hydride reduction of a l iphat ic p r imary

tosyloxy groups r e s u l t s in the replacement of the tosy l ­

oxy group by hydrogen. On the other hand, the reduction

of a secondary tosyloxy group can be expected to undergo

reduct ion to the hydroxyl g roup . If it could be shown

that the th ree p r imary tosyloxy groups of a sucrose to ­

syla te a re reduced to t e rmina l methyl groups by l i thium

aluminum hydr ide , then a means of es t imat ing the num

ber of tosyl groups at the 1 ' - posit ion would be available

s ince the number of tosyloxy groups at the 6- and 6 ' - po-

- 8 2 -

s i t i o n s can be d e t e r m i n e d by i o d i n a t i o n . The r e s u l t s of

the r e d u c t i o n s of s u c r o s e t o s y l a t e and of s u c r o s e m o n o ­

m y r i s t a t e t o s y l a t e a r e given in T a b l e X.

T a b l e X

R e d u c t i o n of the T o s y l a t e s of S u c r o s e and of S u c r o s e Mono­

m y r i s t a t e wi th L i th ium Aluminum H y d r i d e .

T o s y l a t e Solvent T e r m i n a l P e r c e n t C - m e t h y l group y i e ld

e q u i v a l e n t (a)

1. Sucrose 5.67-tosylate diethyl ether 107.8 g. 38

2. " tetrahydrofuran 106.0 g. 38

3. Sucrose mono­myristate 5.24-

tosylate diethyl ether 198.1 g. 85

( a ) . The y i e l d s a r e b a s e d on the a n a l y s e s of the p r o d u c t s .

I t was a s s u m e d tha t the t o sy loxy g r o u p s not r e d u c e d

to t e r m i n a l C - m e t h y l g r o u p s w e r e r e d u c e d to h y d r o ­

xyl f u n c t i o n s . The r e d u c t i o n p r o d u c t s w e r e c h r o m a -

t o g r a p h e d on p a p e r u s i n g the n - b u t a n o l / w a t e r s y s t e m

and the spo t s w e r e l o c a t e d u s ing the p e r m a n g a n a t e -

p e r i o d a t e s p r a y r e a g e n t . Only a few of the compounds

d e t e c t e d by t h i s r e a g e n t ( t h o s e with low Rf v a l u e s )

w e r e s e n s i t i v e to the a n i l i n e p h o s p h a t e s p r a y r e a g e n t .

The r e s u l t s of t h e s e c h r o m a t o g r ams a r e given be low.

- 8 3 -

1. The reduct ion of this tosy la te (see Table VII) gave

four main products having Rf values of 0 .22, 0 .36,

0.45 and 0 . 6 5 . Trea tment of this ma te r i a l with 4 N

hydrochlor ic acid at 80 C. for 15 minutes did not

change the chromatographic sepa ra t ion .

2. This m a t e r i a l on paper chromatograms appeared to

be the same as that obtained in the reduction with

diethyl e ther .

3 . The reduct ion of this tosyla te (sample 3a, Table VII)

gave a mixture of six main components having Rf va­

lues of 0 .074, 0 .135, 0 .235, 0 .34, 0.475 and 0 .662 .

The slower component had an Rf value ident ical to

that of glucose and could be detected by the anil ine

phosphate spray r eagen t .

All the reduct ion products were sulfur free colour

l ess g l a s s e s . At leas t some of the components possessed

r a t h e r high vapour p r e s s u r e s since losses were obtained

in a t tempts to dry the substances under vacuum.

Examination of Table X is sufficient to show that

the reac t ion is very complex. It is in te res t ing to note

that the reduct ion of suc rose 5 . 67 - tosy la te under two dif­

ferent conditions gave ident ica l yields of products which

analyzed very c losely to t r ideoxy sucr os e (C-methyl group

- 8 4 -

equivalent for 6 , 1 ' , 6 ' - t r ideoxy sucr os e is 98.1 g . ) . The

ana lys i s of the product obtained on the reduction of the

suc rose monomyris ta te tosy la te (sample 3, Table X) sug­

ges t s that 1.6 t e rmina l methyl groups were p r e s e n t . The

t heo re t i ca l t e rmina l C-methyl group equivalent for a d i -

deoxysucrose containing two C-methyl groups is 155.15

grams .

The fact that the chromatographic pa t te rn of t he ­

se products was not changed by t rea tment with minera l

ac id , plus the fact that the reduct ion of the sucrose m y r i s ­

ta te tosy la te gave a component in the product mixture

behaving like glucose on paper chromatograms , tend to

show that the glycosidic bonds were broken during the

course of the i so la t ion of the reduction p roduc t s . That

th is is the case was indicated by the fact that the reduc­

tion of suc rose oc taace ta te under ident ical conditions led

to the i so la t ion of glucose and f ruc tose .

In view of the complexity of the reduction products

and the low yields obtained, the r e s u l t s are not amenable

to i n t e r p r e t a t i o n .

- 8 5 -

CLAIMS TO ORIGINAL RESEARCH

The 1 ' - tosyloxy group of a sucrose tosyla te is not

r ep laceab le by an iodine atom.

A method was es tab l i shed for es t imat ing the pe rcen­

tage subst i tu t ion at the 6- and 6*- posi t ions of a su­

c rose d e r i v a t i v e .

A method was developed to es t imate the formyl group

content of a sucrose e s t e r .

Radioact ive sucrose pa lmi ta te having C at the C 1

posi t ion of the palmitoyl group was p r e p a r e d .

Sucrose monomyr is ta tes were separa ted from sucrose

d i m y r i s t a t e s by c o u n t e r - c u r r e n t d i s t r ibu t ion .

Sucrose was separa ted from sucrose myr i s t a t e s by

ex t rac t ion using n-butanol and five percent aqueous

sodium c h l o r i d e .

Proof was obtained that the sucrose monomyris ta tes

obtained by t r a n s e s t e r i f i c a t i o n have approximately

50 percen t of the acyl groups on the 6- and 6 ' - pos i ­

t ions .

A modification of the anil ine phosphate spray reagent

of Bryson and Mitchel l (68) .

A mixture of s u c r o s e , sucrose monoacetates and su­

c rose d iace ta tes was separa ted by par t i t ion chroma­

tography both on paper and on C e l i t e .

- 8 6 -

The acylat ion of sucrose with myr is toyl chlor ide in

pyr idine gives a mixture of highly subst i tu ted sucrose

m y r i s t a t e s .

The l i thium aluminum hydride reduction of sucrose

5 . 67 - to sy la t e gave a mixture of four compounds.

The l i thium aluminum hydride reduction of sucrose

monomyr is ta te 5 .24- tosy la te p repared by t r a n s e s t e r i ­

f ication gave a mixture of at l eas t six compounds.

- 8 7 -

BIBLIOGRAPHY

1 . B . H e l f e r i c h and H . B r e d e r e c k , A n n . 4 6 5 , 1 6 6 , ( 1 9 2 8 ) .

2 . S . V . Shah and Y . M . C h a k r a d e o , C u r r e n t S c i . V o l . 4 , 6 5 2 , ( 1 9 3 6 ) .

3. I. Levi and C.B. Purves, Adv. Carbohydrate Chem., Vol. 4, 1, (1949), Academic Press.

4. W.Z. Hassid and M. Doudoroff, Adv. Carbohydrate Chem. Vol 5, 29, (1950), Academic Press.

5. C.A. Beevers and W. Cochran, Proc. Roy. Soc. (London) A190, 257, (1947).

6 . R . U . L e m i e u x and G . H u b e r , J . A m . C h e m . S o c . 7 5 , 4 1 1 8 , ( 1 9 5 3 ) . J . A m . C h e m . S o c . 7 8 , 4 1 1 7 , ( 1 9 5 6 ) .

7 . R . U . L e m i e u x and J . P . B a r r e t t e , In p r e p a r a t i o n .

8 . G . G . M c K e o w n , R . S . E . S e r e n i u s and L . W . H a y w a r d ,

C a n . J . C h e m . 35 , 2 8 , ( 1 9 5 7 ) .

9 . R . C . H o c k e t t , J . A m . C h e m . S o c . 7 2 , 1 8 3 9 , ( 1 9 5 0 ) .

1 0 . F . D - S n e l l , I n d . E n g . C h e m . 3 5 , 1 0 7 , ( 1 9 4 3 ) .

1 1 . E . K . F i s c h e r , S o a p , S a n i t . C h e m i c a l s , 1 9 , 2 2 5 , ( 1 9 4 3 ) .

1 2 . C . S c h o e l e r and M . W i t t w e r , U . S . p a t . 1 , 9 7 0 , 5 7 8 , ( 1 9 3 4 ) .

1 3 . A . T . B a l l u m , J . M . S c h u m a c h e r , G . E . K a p e l l a and J . V . K a r a b i n o s , J . A m . Oi l C h e m i s t ' s S o c . 3 1 ,

2 0 , ( 1 9 5 4 ) .

1 4 . J . V . K a r a b i n o s and M . C . M e t z i g e r , T r a n s a c t i o n s of I l l i n o i s A c a d e m y of S c i e n c e , 4 8 , 1 1 8 , ( 1 9 5 6 ) .

1 5 . M . B e r t h e l o t , C o m p t . r e n d . 4 1 , 4 5 2 , ( 1 8 8 5 ) . A n n . c h i m . p h y s . 6 0 , 9 3 , ( 1 8 6 0 ) .

1 6 . I . B e l l u c i , G a z z . c h i m . i t a l . , 42JI> 2 8 3 , ( 1 9 1 2 ) .

1 7 . T . P . H i l d i t c h and J . G . R i g g , J . C h e m . S o c . 1774 ( 1 9 3 5 ) .

- 8 8 -

1 8 . J . P . G i b b o n s and R . A . J a n k e , J . A m . O i l C h e m i s t ' s S o c . 2 9 , 4 6 7 , ( 1 9 5 2 ) .

1 9 . C . J . C a r r a n d J . C . K r a n t z , A d v . C a r b o h y d r a t e C h e m . V o l . 1, 1 8 0 , ( 1 9 4 5 ) .

2 0 . W . R . B l o o r J . B i o l . C h e m . 7 , 4 2 7 , ( 1 9 1 0 ) . 11 , 141 and 4 2 9 , ( 1 9 1 2 ) .

2 1 . K . R . B r o w n , U . S . p a t . 2 , 3 2 2 , 8 2 0 , ( 1 9 4 3 ) . 2 , 3 2 2 , 8 21, 2 , 3 2 2 , 8 2 2 ,

2 2 . K . H e s s and E . M e s s m e r , B e r . 5 4 , 4 9 9 . ( 1 9 2 1 ) .

2 3 . W. C a r p m a e l , B r i t . p a t . 2 3 9 , 7 2 6 , ( 1 9 2 4 ) .

2 4 . A . J . F a r b e n i n d , G e r . p a t . 4 7 8 , 1 2 7 , ( 1 9 2 4 ) .

2 5 . A . G . G o l d s m i t h , F r e n c h p a t . 6 6 4 , 2 6 1 , ( 1 9 2 9 ) .

2 6 . L . R o s e n t h a l and W. L e n h a r d , U . S . p a t . 1 , 7 3 9 , 8 6 3 , ( 1 9 2 9 ) , G e r . p a t . 4 1 1 , 9 0 0 , ( 1 9 2 5 ) .

2 7 . B . J . H a r r i s , U . S . p a t . 1 , 9 1 7 , 2 5 0 , ( 1 9 3 3 ) .

1 , 9 1 7 , 2 5 7 ,

2 8 . E . J . L o r a n d , U . S . p a t . 1 , 9 5 9 , 5 9 0 , ( 1 9 3 4 ) .

2 9 . J . O . C l a y t o n , E . G . S t u a r t and F . A . S t u a r t , U . S . p a t .

2 , 7 0 0 , 0 2 2 , ( 1 9 5 5 ) .

3 0 . C h e m i c a l a n d E n g i n e e r i n g N e w s , J u n e 3 , 9 0 , ( 1 9 5 7 ) .

3 1 . A . G r u n , F . W i t t k a and J . S c h o l z e , B e r . 5 4 , 2 9 0 , ( 1 9 2 1 ) .

3 2 . H . J . W r i g h t , J . B . S e g u r , H . V . C l a r k , K . S . C o b u r n , E . E . L a u g d o m and R . N . D u P u i s , O i l and S o a p , 21 ,

1 4 5 , ( 1 9 4 4 ) . 3 3 . A . T . G r o s s a n d R . O . F e w g e , J . A m . O i l C h e m i s t ' s

S o c . _26, 7 0 4 , ( 1 9 4 9 ) .

3 4 - H . S . G i l c h r i s t , R e p t . B r i t . A s s o c . A d v a n c e m e n t S c i . 3 5 7 , ( 1 9 2 2 ) .

3 5 . A . L a p w o r t h and L . K . P e a r s o n , B i o c h e m J . 1 3 , 2 9 6 , ( 1 9 1 9 ) .

- 8 9 -

H . B u r r e l l . O i l and S o a p , 2 1 , 2 0 6 , ( 1 9 4 4 ) .

J . C . I r v i n e and H . S . G i l c h r i s t , J . C h e m . S o c . .125, 1, ( 1 9 2 4 ) .

H . Wolff and W . H . H i l l , J . A m . C h e m . S o c . 2 5 , 2 5 8 , ( 1 9 4 8 ) .

J . C . K o n e n , E . T . C l o c k e r a n d R . P . C o x , O i l and S o a p , 2 2 , 5 7 , ( 1 9 4 5 ) .

H . A . G o l d s m i t h , C h e m . R e v i e w s , 33_, 2 5 7 , ( 1 9 4 3 ) .

F . D . S n e l l , L . O s i p o w , W . C . Y o r k and A . F i n c h l e r , I n d . E n g . C h e m . 4 8 , 1 4 5 9 , ( 1 9 5 6 ) .

F . D . S n e l l , and L . O s i p o w , C h e m . P r o d u c t s , 2 0 , 1 0 1 , ( 1 9 5 7 ) .

F . D . S n e l l , P r i v a t e c o m m u n i c a t i o n , June 5 t h , ( 1 9 5 5 ) .

F . D . S n e l l and C . T . S n e l l , C o l o r i m e t r i c M e t h o d of A n a l y s e s , V o l . 3 , 4 5 , ( 1 9 5 3 ) .

Van N o s t r a n d , N . Y .

F . D . S n e l l , L . O s i p o w , W . C . Y o r k and D . M a r r a , I n d . E n g . C h e m . 4 8 , 1 4 6 2 , ( 1 9 5 6 ) .

C . Z . D r a v e s and R . G . C l a r k s o n , A m . D y e s t u f f R e p t r . 2 0 , 2 0 1 , ( 1 9 3 1 ) .

J . R o s s and G- M i l e s , Q i l and S o a p , 18_, 9 9 , ( 1 9 4 1 ) .

J . C o u r t o i s and M . R a m e t , C o m p t . r e n d . 21_^, 3 6 0 , ( 1 9 4 9 ) , B u l l . s o c . c h i m . h i o l . - , 27_, 6 1 0 , ( 1 9 4 5 ) .

J . A s s e l i n e a u , B u l l . s o c . c h i m . F r a n c e , 9 3 7 , ( 1 9 5 5 ) .

R . S . T i p s o n , A d v . C a r b o h y d r a t e C h e m . V o l . 8 , 1 0 7 , ( 1 9 5 3 ) . A c a d e m i c P r e s s .

A . B e r n o u l l i and H . S t a u f f e r , H e l v . C h i m . A c t a , 2 3 , 6 1 5 , ( 1 9 4 0 ) .

J . C o m p t o n , J . A m . C h e m . S o c . 6 0 , 3 9 5 , ( 1 9 3 8 ) .

R . C . H o c k e t t and M . L . D o w n i n g , J . A m . C h e m . S o c , 6 4 , 2 4 6 5 , ( 1 9 4 2 ) .

- 9 0 -

F . A . M e n a l d a , R e c t r a v . c h i m . 4 9 , 9 6 7 , ( 1 9 3 0 ) .

R . C . H o c k e t t and M . Z i e f , J . A m . C h e m . S o c , 7 2 , 1 9 3 9 , ( 1 9 5 0 ) .

J . W . O l d h a m a n d J . K . R u t h e r f o r d , J . A m . C h e m . S o c . 5 4 , 3 6 6 , ( 1 9 3 2 ) .

R.S. Tipson and P. Black, J. A m . Chem. Soc, 66, 1880, (1944).

A.L. Raymond and E.F. Schroeder, U.S. pat. 2,365,776, (1944).

C.H. Hudson, R.M. Hann and A.T. Ness, J. A m . Chem. Soc, 66, 73, (1944).

D . J . B e l l , E . F r i e d m a n n and S . W i l l i a m s o n , J . C h e m . S o c , 2 5 2 , ( 1 9 3 7 ) .

F . D . S n e l l , A . F i n c h l e r , W . C . Y o r k and L . O s i p o w ,

J . A m . O i l C h e m i s t ' s S o c . 33_, 4 2 2 , ( 1 9 5 6 ) .

S . T . B a u e r , O i l and S o a p , 2 3 , 1, ( 1 9 4 6 ) .

E . M a t t h e r and H . Z i e g e n s p e c k , B o t . A r c h i v . , _15_, 1 8 7 , ( 1 9 2 6 ) .

J . M i t c h e l l J r . , D . M . S m i t h , and F . S . M o n e y , I n d . E n g . C h e m . A n a l . E d . , 1_6,

4 1 0 , ( 1 9 4 4 ) .

W . M . G r a n t , A n a l . C h e m . 2 0 , 2 6 7 , ( 1 9 4 8 ) .

M . L a m b e r t and A . C . N e i s h , C a n . J . R e s e a r c h , B2JJ, 8 3 , ( 1 9 5 0 ) .

F . S m i t h and M . A b d e l A k h e r , J . A m . C h e m . S o c . , 7 3 , 5 8 5 9 , ( 1 9 5 1 ) .

J . L . B r y s o n and T . J . M i t c h e l l , N a t u r e , 1^67, 8 6 4 , ( 1 9 5 1 ) .

R . U . L e m i e u x and H . F . B a u e r , A n a l . C h e m . , 2 6 , 9 2 0 , ( 1 9 5 4 ) .

S . M . P a r t r i d g e , N a t u r e , ^ 6 4 , 4 4 3 , ( 1 9 4 9 ) .

- 9 1 -

7 1 . R . U . L e m i e u x , C . T . B i s h o p and G . E . P e l l e t i e r .

C a n . J . C h e m . , 3 4 , 1 3 6 5 , ( 1 9 5 6 ) .

7 2 . D . L . M o r r i s , S c i e n c e , 1 0 7 , 2 5 4 , ( 1 9 4 8 ) .

7 3 . K . H . M e y e r , O r g a n i c S y n t h e s e s , C o l . V o l . 1, 6 0 .

7 4 . R . U . L e m i e u x and H . F . B a u e r , C a n . J . C h e m . , 3 2 , 3 4 0 , ( 1 9 5 4 ) .

7 5 . W . H . M c N e e l y , W . W . B i n k l e y and M . L . W o l f r o m , J . A m . C h e m . S o c , 6 7 , 5 2 7 , ( 1 9 4 5 ) .

7 6 . R . S . T i p s o n , A d v . C a r b o h y d r a t e C h e m . V o l . 8 , 1 0 7 , ( 1 9 5 3 ) . A c a d e m i c P r e s s .

7 7 . O . E . S u n d b e r g and G . L . R o g e r , I n d . E n g . C h e m . A n a l . E d . , 1 8 , 7 1 9 , ( 1 9 4 6 ) .

7 8 . J . M . B o b b i t , A d v . C a r b o h y d r a t e C h e m . V o l . 1 1 , 1 , ( 1 9 5 6 ) . A c a d e m i c P r e s s .

7 9 . P . K a r r e r and H . S c h m i d , H e l v . C h i m . A c t a , 3 2 , 1 3 7 1 , ( 1 9 4 9 ) .

8 0 . R . U . L e m i e u x and C . B . P u r v e s , C a n . J . R e s e a r c h ,

B 2 5 , 4 8 5 , ( 1 9 4 7 ) .

8 1 . L . O s i p o w , P r i v a t e c o m m u n i c a t i o n , J u n e 2 7 t h , ( 1 9 5 5 ) .

8 2 . F . A r n d t , O r g a n i c S y n t h e s e s , C o l . V o l . 2 , 1 6 5 , John W i l e y and S o m s .

8 3 . P . B . D . de l a M a r e , L . F o w d e n , E . D . H u g h e s , C . K . I n g o l d and D . H . M a c k i e , J . C h e m . S o c . ( 1 9 5 5 ) , 3 2 0 0 .

8 4 . A . J . L i s t o n , U n p u b l i s h e d w o r k , t h i s l a b o r a t o r y .

8 5 . K . M . H e r s t e i n , P r i v a t e c o m m u n i c a t i o n .

8 6 . J . M . S u g i h a r a , A d v . C a r b o h y d r a t e C h e m . V o l . 8 , 1 , ( 1 9 5 3 ) , A c a d e m i c P r e s s .

8 7 . P . K a r r e r a n d A . K . M i t r a , H e l v . C h i m . A c t a , 3 8 , 1, ( 1 9 5 5 ) .