2075 Wesbrook Place. Vancouver, Canada. V 6 T 1W5. Date ...... H. Thurow, Y.M. Choy, N. Frank, H. Niemann , and S. Stirm. Carbohydr. Res.,41,241-255 ( 1 9 7 ...
STUDIES ON BACTERIAL CAPSULAR POLYSACCHARIDES AND ON A PLANT GUM
by JOSE LUIS LDI FABIO B.Sc., U n i v e r s i d a d de l a R e p u b l i c a , Uruguay, 1 9 7 5
A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE
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in THE
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The U n i v e r s i t y o f B r i t i s h 2075 Wesbrook P l a c e Vancouver, Canada V 6 T 1W5
Date
i?/7
>
Figure II.1
M.s.
o f (a)
1,2,5-tri-0-acetyl-3»k,6-tri-0-
methyl g a l a c t i t o l
and
(b)
-3-0-ethyl-4,6-di-0-methyl
1,2,5-tri-O-acetyl galactitol.
3k The
primary fragments formed g i v e r i s e t o secondary f r a g -
ments by l o s s o f a c e t i c a c i d ol
(M.W.
(M.W.
60),ketene
(M.W.
42),methan-
3 2 ) or formaldehyde (M.W. 3 0 ) .
T h i s i n f o r m a t i o n was used t o i d e n t i f y s p e c i f i c compounds f o r which standard s p e c t r a
were not a v a i l a b l e . F o r
example,when
u r o n i c a c i d d e g r a d a t i o n was performed on K l e b s i e l l a K 6 0 p o l y s a c c h a r i d e , the
p o s i t i o n where the u r o n i c a c i d was a t t a c h e d was
l a b e l l e d w i t h e t h y l i o d i d e . T h e e t h y l a t e d , p a r t i a l l y methylated a l d i t o l a c e t a t e thus obtained
(
l,2,5-tri-0-acetyl-3-0-ethyl-
4 , 6 - d i - 0 - m e t h y l g a l a c t i t o l ) was i d e n t i f i e d by means o f i t s m.s. and comparison w i t h the c o r r e s p o n d i n g methylated tive
(l,2,5-tri-0-acetyl-3t4,6-tri-0-methyl
galactitol).Several
masses are s h i f t e d by 14 u n i t s as i l l u s t r a t e d The
f r a g m e n t a t i o n expected i s shown below:
deriva-
i n F i g u r e II.1.
35
Primary f r a g m e n t a t i o n
CHgOCOCH^
CH ,0C0CHo I2 3
CH-OCH,
CH-0CH CH~ + 2 3 (m/e 203)
CHOCOCH^
I
CHOCOCH^ O
CHgOCH^ (m/e 161)
CHOCH, :H=OCH CH
CHOCOCHI 3 CH 0CH £
3
2
CHgOCOCH^
3
HOCOCH^
3
;HOCOCH
IH 0CH 2
;HOCH CH 2
3
CH-OCH+ 3 (m/e 2 4 7 )
3
3
(m/e 219)
Secondary f r a g m e n t a t i o n
CH 0CH 2
CHOCH-
3
CH0C0CH" CH=0CH
-Ac OH
3
II
H
3
H=0CH
3
3
(m/e 101)
(m/e 161)
CH, II
-Me OH
z
-CH C0 2
HOCOCH,
o
-ArOH Z^im
3
CH=0CH CH 2
(m/e
3
203)
CHOCOCH" CH CH=0CH CH 2
(m/e 1 4 3 )
3
(m/e
(m/e 129)
2
4= 0 CH=0CH
H-OCH^
H 0C0CH^ 3 H0C0CH
CH
3
3
87)
36 II.5 Sequencing o f sugars. The
i s o l a t i o n and c h a r a c t e r i z a t i o n o f fragments o f a p o l y -
saccharide
are the keys t o the d e t e r m i n a t i o n
o f the s e q u e n t i a l
arrangement o f the c o n s t i t u e n t monosaccharides i n the polymer. By u t i l i z i n g d i f f e r e n t techniques i t i s p o s s i b l e t o o b t a i n these fragments and by d e t e r m i n i n g the r e l a t i v e p o s i t i o n s o f one
t o the o t h e r , i n an a d d i t i v e manner,is p o s s i b l e t o b u i l d up
the sequence i n the p o l y s a c c h a r i d e
or i n the r e p e a t i n g u n i t .
II.5.1 P a r t i a l h y d r o l y s i s " ^ . 8 2
A considerable
8
amount o f data on r a t e constants
and k i n e t i c
parameters f o r the a c i d c a t a l y z e d h y d r o l y s i s o f g l y c o s i d e s o f 1
82 83 monosaccharides has been presented i n s e v e r a l reviews
' . J
Many f a c t o r s seem t o i n f l u e n c e the r a t e o f h y d r o l y s i s , such as r i n g size,configuration,conformation,polarity
o f the sugar ,
s i z e and p o l a r i t y o f the aglycon.Although data on
polysaccha-
r i d e s are not so e a s i l y a v a i l a b l e . i n f e r e n c e s can be made from the r e s u l t s on the monosaccharide g l y c o s i d e s , a n d
may be sum-
marized as f o l l o w s : i)
furanosides
are more l a b i l e than
pyranosides,
i i ) deoxysugars are more e a s i l y hydrolyzed
than hexoses,
i i i ) pentopyranosides a r e more a c i d l a b i l e than hexopyranosi des, iv)
a - g l y c o s i d e s are g e n e r a l l y more l a b i l e than 3 - g l y c o s i d e s ,
v)
(1-6) l i n k a g e s are more r e s i s t a n t t o a c i d h y d r o l y s i s than
(1-2),(1-3) and (1-4) v i ) residues hydrolyzed
present
linkages,
on s i d e chains
than when present
are o f t e n more e a s i l y
i n the main c h a i n ,
37 v i i ) u r o n i c a c i d s are more r e s i s t a n t to h y d r o l y s i s , v i i i ) aminosugars are more a c i d r e s i s t a n t than common hexoses. In h e t e r o p o l y s a c c h a r i d e s
we
f i n d t h a t some g l y c o s i d i c
l i n k a g e s are more r e s i s t a n t than others,so
t h a t , under c e r t a i n
c o n d i t i o n s of h y d r o l y s i s ( a c i d concentration,temperature time of h y d r o l y s i s ) s e l e c t i v e cleavages polysaccharide polysaccharides
w i l l occur
and
i n the
y i e l d i n g defined oligomeric units.For a c i d i c c o n t a i n i n g u r o n i c a c i d s .there s i s tance of
the
u r o n o s y l bond l e a d s to the accummulation of a l d o b i o u r o n i c and
t o a l e s s e r extent
acid
the a l d o t r i o u r o n i c and/or a l d o t e t r a o -
u r o n i c a c i d fragments.In p o l y s a c c h a r i d e s
w i t h deoxysugars i t
i s d i f f i c u l t to i s o l a t e , b y p a r t i a l h y d r o l y s i s oligomers w i t h deoxyhexosyl bonds.Usually a f t e r a p a r t i a l h y d r o l y s i s , t h e amount of monosaccharides i s high.By c o u p l i n g ' s e v e r a l t i o n techniques
i t i s p o s s i b l e to separate
separa-
the oligomers from
the monosaccharides as w e l l as to i s o l a t e pure o l i g o s a c c h a r i des. The
techniques
most commonly used are paper chromatogra-
phy ,gel-permeation chromatography,paper e l e c t r o p h o r e s i s g a s - l i q u i d chromatography (see S e c t i o n Because a c e t a l s . a c e t a t e s and i n the p o l y s a c c h a r i d e s bonds.it
and
II.5.*0.
formates t h a t may
be
present
are more a c i d l a b i l e than g l y c o s i d i c
i s o f t e n p o s s i b l e to s e l e c t i v e l y remove these sub-
s t i t u e n t s without a f f e c t i n g the r e s t of the
polysaccharide.
P a r t i a l h y d r o l y s i s can a l s o be c a r r i e d out on f u l l y methylated
polysaccharides. A complementary technique i s a c e t o l y s i s - \ a s the 8
r a t e of cleavage of the g l y c o s i d e s i n the two
relative
mechanisms i s
38 reversed.During cleaved,while II.5.2
acetolysis,(1-6)
they
Periodate
a r e t h e most
oxidation
linkages stable
and Smith
are
preferentially
during
acid
hydrolysis.
h y d r o l y s i s ^ " ^ .
86 It salts give
has been
known s i n c e
are capable two
1928
that
a,B-glycols
of cleaving
H
ICv"
H
CHOH
C
^
to
H
+ H„0 + 10 ~
0
^
J
^2
When more tions, formic
than acid
two h y d r o x y l groups i s
are i n contiguous
posi-
released,
R,
R,
I CHOH I (j)H0H
I
1
+
210^
+HC0 H + H 0+ 2 I 0 2
R
one o f
the v i c i n a l
h o l , formaldehyde
C H OH
A This
i s
produced,
+
i s
a primary
alco-
HCHQ
I OK
CHO
+
H 0 ?
+
10
R reaction
carbohydrate formation
termined
3
2
hydroxyl groups
2
CHOH
2
CHO
*2 When
1
CHO
CHOH
a
quantitatively
CHO
^2
as
and i t s
J l 0
+
the
acid
aldehydes:
?1 C
periodic
has found
chemistry.Since of formic
accurately
an analytical preparative
a wide
acid
range
of applications
the reduction
of periodate
and formaldehyde
on the microscale,periodate
technique,but
procedure.
i n a d d i t i o n . i t
i n and
c a n a l l be d e i s
invaluable
c a n be used
as
39 When of
used
as
an
polysaccharide
a n a l y t i c a l
is
oxidized
oxidation
being
periodate
and
i f
formed.Over-,and
any
is
plicate the
monitored
the
release
and
at
low
repulsions
charide
in
i f
certain
sugar 91
hemiacetals and
problem i t
the
of
formic
free is
the
hydroxyl
overcome
by
subjecting
oxidation
is
hydrolysis
the
gives
r e s i s t a n t
tent
with
the
possible
the
by
the
may
the
periodate,the
and/or may
occur
due
com-
working
i n
solutions to
of
electrothe
polysac-
a f f e c t s
both
periodate
ions
hindrance
formation
however
and
the
of
formaldehyde
by
ions
to
amount
consumption
buffered
d i o l s
known
of
minimized
periodate
dialdehydes
groups
i n
a d d i t i o n
r e p u l s i o n
them
to
very
sugars
products of
of
intramolecular
requisite
polysaccharide
are
of
not
(NaClO^)
be
exposed
the
products
i n
polysaccharide
data.As
formed
that
and
and
an
used
some
of
oxidized
by
oxidation
of
as the
a
the
released
number should
upon
of be
periodate
monosubstituted
is
to
p e r i o consis-
II.2
periodate
shows
consumed
hexoses.
fragmentation
sugar
inhib-
protected
example.Figure
moles
f i r s t
reduction.When
information.The
the
oxida-
which
substrate.The
borohydride
oxidation is
salts
can
by
polysaccharide.The
the
of
generated
u s e f u l
terminal
periodate
the of
hemiacetals)
methylation
oxidation
n i q u e , the
acid
with
7
complete.analysis
date
When
is
v i c i n a l
through
7
e l e c t r o s t a t i c
f i r s t
the
under-oxidation
residues,and
(with
upon
measuring
,a
92 '
7
of
residues
the
by
oxidation
between
87 88
solution
acidic;furthermore,steric
a c c e s i b i l i t y
tion
a
temperatures
periodate.Incomplete
the
i n
results.Overoxidation*^
dark
s t a t i c
technique
residues and
can
be
techi n
a
se-
40
Products
T e r m i n a l a n d monosubstituted
formed a f t e r o x . and
hexoses
hydrolysis CHnOH *-
'
CHO
h-OH + I HO
C^OH
OH
CHUOH
j
CHO
1
hOH • CH^DH CHJOH
CH^DH
—OH
- O H
-OH
HO-
CH-OH
IHJOH
CHJDH HjOH
V-C—
CH^H HO—/
*
cr
CHjOH
HO—/
CHO
CHJOH
CHO
UoH + h-OH
\ — 0 -
CHO HOH
tHJDH CH-DH
CH^H QL
F i g u r e I I . 2 . Common p r o d u c t s tion
f o r m e d on p e r i o d a t e o x i d a -
o f t e r m i n a l and m o n o s u b s t i t u t e d
hexoses.
41
parated
from the o x i d i z e d r e s i d u e s as mono-,oligo- or p o l y -
s a c c h a r i d e d e r i v a t i v e s a f t e r some chemical m o d i f i c a t i o n s . I f , a f t e r oxidation with periodate,the
aldehydes generated are
reduced t o a l c o h o l s w i t h sodium borohydride,the ( p o l y o l ) thus obtained
polyalcohol
i s c o n s t i t u t e d by g l y c o s i d i c l i n k a g e s 03
as w e l l as by a c y c l i c a c e t a l groupings.Smith
olf
degradation ^ 7
i s based on the d i f f e r e n t s t a b i l i t y towards a c i d of the g l y c o s i d i c bond and an a c y c l i c acetal.The
l a t t e r hydrolyzes
much
f a s t e r . T h e p o l y o l i s t r e a t e d w i t h m i l d a c i d and the products obtained,comprising;
a) s m a l l fragments d e r i v e d from o x i d i z e d ,
r e s i d u e s , o r b) g l y c o s i d e s o f mono- or o l i g o - s a c c h a r i d e s (the aglycons
being the s m a l l fragments mentioned b e f o r e ) and p o l y -
s a c c h a r i d e s d e r i v e d from non-oxidized
fragments are i s o l a t e d
and p u r i f i e d by d i f f e r e n t s e p a r a t i o n techniques I I . 5 . 4 ) and c h a r a c t e r i z e d by c o n v e n t i o n a l
(see S e c t i o n
procedures.Based
on the d i f f e r e n c e s i n the r a t e s o f o x i d a t i o n of v i c i n a l
diols
( c i s being o x i d i z e d f a s t e r than t r a n s ) , t h e f a c t t h a t t e r m i n a l r e s i d u e s are more a c c e s s i b l e t o p e r i o d a t e uronate anions tend t o r e p e l l the p e r i o d a t e
ions and t h a t
i o n s . i t i s pos-
s i b l e to s e l e c t i v e l y oxidize c e r t a i n residues
i n the p o l y s a c -
c h a r i d e ^ , doing what may be c a l l e d a " s e l e c t i v e Smith degradation" . 9 6
Scheme I I . 2 shows the Smith degradation K 26
of K l e b s i e l l a
polysaccharide.
II.5.3 Degradations based on g - e l i m i n a t i o n ' 7
.
S e v e r a l groups ( a l k o x y l , h y d r o x y l , e t c . ) i n the B - p o s i t i o n to an e l e c t r o n withdrawing group,such as c a r b o n y l , c a r b o x y l i c
kz
1. NalO^ 2. (CH OH) 3. NaBH 2
2
4
Z>. 0.5MTFA r.t. 24h 5. NaBty
Scheme 11,2
Smith
d e g r a d a t i o n o f K l e b s i e l l a K 26
polysaccharide.
*3 ester,amide or s u l f o n e are e l i m i n a t e d on treatment w i t h "base. The
presence of a hydrogen atom i n the 2
6 4.42
A^ was
2Hz),
6 4.87
(1.0H,J
1
2
( O ^ H . J ^ 8 H z ) and 6 2
showed s i g n a l s a t 6
2
(0.6H,J
8Hz).
o f A^ showed s i g n a l s
6
lj2
7 H z ) , 6 4.52
(see Table V . 3
4.52 5-25
(1.0H,J
lf2
7Hz)
and Spectrum N°30).
i d e n t i f i e d as 6-0-( R -D-glucopyranosyl u r o n i c a c i d ) - D -
g a l a c t o s e and
the i d e n t i t y confirmed by co-chromatography w i t h
an a u t h e n t i c sample. A
2
was
i d e n t i f i e d as 6-0-( g-D-glucopyran-
o s y l uronic a c i d ) - 6 - 0 - ( 8 -D-galactopyranosyl)-D-galactose.
174
( 1 0 mg),Ng ( 1 5 mg) and
Three n e u t r a l o l i g o s a c c h a r i d e s ,
( 5 mg) were i s o l a t e d by p r e p a r a t i v e paper chromatography from the n e u t r a l f r a c t i o n . A n a l y s i s from these i n Table
V . 2 , i n d i c a t e d the s t r u c t u r e s shown belows
N-^ 3 - 0 - ( 3 N
2
oligosaccharides,given
6-0-(
-D-galactopyranosyl)-D-galactose, B-D-galactopyranosyl)-D-galactose,
N^ 3-0-( B-D-galactopyranosyl)-6-0-(
g
-D-galactopyranosyl)-D-
galactose. A sample o f the gum
( 2 5 0 mg) was t r e a t e d w i t h
I M TFA on
a steam-bath f o r 1-f h.The a c i d was removed and the r e s i d u e d i s s o l v e d i n water ( 1 0 mL) and d i a l y z e d f o r 7 2 h a g a i n s t l e d water (l.OL).The +20°
non-dialyzable
6 4 . 5 3 (J^
2
distil-
m a t e r i a l ( 5 0 mg) h a d [ a ] ^
(c 1 . 7 » w a t e r ) and the "hi-n.m.r. spectrum i n DgO
s i g n a l s a t 6 5 - 4 0 and
was
recorded
^Hz) i n a l s l r a t i o . S u g a r
a n a l y s i s gave mannose,galactose and g l u c u r o n i c a c i d i n a r a t i o of 3 - 9 s 1 . 0 : 4 . 1 . M e t h y l a t i o n Table
a n a l y s i s gave the r e s u l t s shown i n
V.l,column I I I . H y d r o l y s i s o f t h i s m a t e r i a l ( 2 0 mg)
with
2M TFA on a steam-bath f o r 7 h showed by p.c. ( s o l v e n t ( A ) ) , galactose,mannose.glucuronic a c i d , t h e a l d o b i o u r o n i c a c i d A^ , the a l d o b i o u r o n i c a c i d A^ and other h i g h e r
oligomers.A^ ( 6 mg)
was i s o l a t e d by paper chromatography and analyzed Table
V . 2 . The ^H-n.m.r. spectrum (see Table
as shown i n
V . 3 and spectrum
N ° 3 D showed s i g n a l s a t 6 5 . 3 0 ( 0 . 8 H , s ) , K22
1
®
K38
K3?
Uronic a c i d i n c h a i n , a) l i n e a r . _ X -
0 - 0 -
K l , K5,
K63
- X - 0 - 0 - 0 -
K4,
_ x
K6
0 - 0
K9*,
_ X - 0 - 0 - 0 - 0 - 0 K70,
-
K81
-
K44
192
b) branch p o i n t on u r o n i c
acid
i ) s i n g l e u n i t side chain _ X - 0 - 0 -
- X - 0 - 0 - 0 -
I
I
0
0
Kll, ii)
K5?
K21, K24
two u n i t s i d e c h a i n
- X - 0 - 0 -
- X - 0 - 0 - 0 -
1
I
0
0
1
I
0
0
K31 i i i ) three u n i t s i d e c h a i n
K46
- X - 0 - 0 - 0 -
I 0
I
0
K26
I
0 i v ) p l u s branch p o i n t s on n e u t r a l sugars X - 0 - 0 - 0 -
I
|
|
0
0
0
K60
c) branch not on u r o n i c _ X - 0 - 0 -
I
0 K58
acid
- x - O - O - O -
- X - 0
I
I
0 K7, K61, K62
0 K52,
193 • - x - 0 - 0 -
•.
- X - 0 - 0 - 0 -
I
I
0
0
K16, K54 d)
double
Kl?
branch
not
on
uronic
acid
0 - X - 0 - 0 - 0 -
I
0 Uronic a)
acid
single
i n
side
chain
unit
side
chain
_ 0 - 0 - 0 -
I
I
X
X
K2, b)
_ o - 0 - 0 - 0 -
two
K8 single
K9t unit
side
K59
chain
- 0 - 0 - 0 - 0 - 0 -
I
I
X
0
K
c)
two
single
1
. 0 - 0 - 0 -
location
chains
r , j
units
0
exact
side
chain
forming
a
not
I
- 0 - 0 - 0 - 0 -
I
I
X
X
K30.K33
K27
side
determined
double
0
of
branch
194
d) two u n i t s i d e c h a i n i)
uronic acid terminal - 0 - 0 -
I K20, K23, K51. K55
0
X
i i ) uronic a c i d non-terminal - 0 - 0 -
- 0 - 0 - 0 -
I
- 0 - 0 - 0 - 0 -
I
X
X
I
0 K25, K47
I X
I
I
0 K13. K74
0 K12, K28, K36
e) three u n i t s i d e c h a i n i ) uronic a c i d non-terminal - 0 - 0 - 0 -
- 0 - 0 - 0 - 0 -
I
I
0
X
1 X
I
I 0
I
0
0
K18
K4l
195
APPENDIX I I THE STRUCTURES OF KLEBSIELLA CAPSULAR POLYSACCHARIDES
196
Structure
K-type Kl
1 man— 3
K2
a
1 G].cA K4
-2G1C—^GlcA-—^Man^—2(ji I_ c
P
K5
-AucA 1
4
2
A
V
1
1
B
OAc
K6
A
-2-Fuc-—^Glc-—^anl—^1 A— C
a
B
B
a
Gal ll
K7
P
4 6—2
1
^ Gal^OH B
^C n.m.r. 20 MHz, amb.temp.
acetone 31 . 0 7
I ' I
1
I
1
I
1
1
I
1
1
1
I
1
I
1
I
1
i
'
i
'
i
' i
I
K 60 Compound N
Spectrum No. 5
1
Glcv/OH
Glc ^rr- Man
13 C
n.m.r*
20 MHz. amb.temp.
102.54
99.85
96.80 83
93.07
K
60,
P
hal-
[ -JGICAJ 8 1„ H
Spectrum
polysaccharide
1
2 -
^Glc—]
Man
a
a
B
n
n.m.r.
100 MHz, 90°C
5.27
5.33
No.6
K 60, P-| p o l y s a c c h a r i d e y
Z
Spectrum
No.?
n.m.r.
2 0 MHz, amb.temp.
acetone 31 .07
101 . 4 2
Spectrum No. 11
K 60 , Compound GlcA-} ^
H
^QalB
^MarJ a
2G1C~0H a
n.m.r.
100 MHz,
90° C
5. 5.31
K 60, Compound A^
Spectrum No.12
^ C n.m.r. 2 0 MHz, amb.temp.
ro ro 101.51 101.35
104.51
96.67 I
I
93
1
K 26 , depyruvylated
polysaccharide
Spectrum No. 1 3
1
H
n.m.r
400 MHz
, water n u l l acetone 2.23
4.51 5.^9
5.rTs-01 4 . 6 3 [ 4 . 4 5
K 26,Compound
GlcA - — ^ Man~OH
1H n.m.r. a
100 MHz, 90°C
5.32 5.20
4.92
»)0M^
-J
1 1 1 1 1
1
1 1 1 1
I I I I
' '_i J 1 1 1
i rJ
,
1 1
L_l
K 26,Compound
Spectrum No.15
GlcA - — ^ Man~OH 13C J
° n.m.r.
20 MHz, amb.temp.
acetone 31.07
K 26,Compound
GlcA - — ^ a
M
a
n
A
Spectrum No.l?
2
1—?. Man^OH a
n.m.r. 2 0 MHz , amb.temp. 102.86
101.36
acetone 31.0?
93.56 93-35
i ' i
1
i ' I
1
I
1
I
r-r-' | i |
ro ro vo
Spectrum No.19
K 26, Compound Aj GlcA - — 2 .
M
a
n
1—2
a
M
a
n
1—1 Gal~OH
a
a
13 -^C n.m.r. 20 MHz , amb.temp.
'
I
'
I
1
I
1
l
1
l
1
I
1
I
1
1
1
I
1
I
1
I
r
K 26, Compound N Glc — Glc~OH 1 H n.m.r. 8
100 MHz ,90°C
Spectrum No.21
K 26 .Compound 1^ Glc - — - Glc~OH B
1 3' rC
n.m.r.
2 0 MHz , amb.temp.
61.60
acetone 31*0?
ro
103.50
96.81
92.95
K 26,Compound N
Spectrum No.22
2
Gal - — - Glc - — - Glc~OH *H n.m.r. 400 MHz , amb.temp.
HOD
4.46
acetone 2.23
4.5 5.24
4.67*
I
Spectrum No.23
Spectrum No. 24
K 26, Compound SH Gal
GlcA — ^ Man — - Gly 1
8
a
a
^C n.m.r. 20 MHz ,amb.temp.
acetone 31.07
0 6 0 K60, Compound X
Spectrum No.25
^C n.m.r.
20 MHz,amb.temp.
acetone 31.07
0 6 0 K60 , Compound P
n -^C n.m.r. 20 MHz
,amb.temp.
2
Spectrum NoZ6
046 X
K46,Compound
?
Spectrum No.2?
1
H n.m.r.
400 MHz, water n u l l acetone 2.23
ro
V>)
1.52
046
K46,Compound
P
x
Spectrum No.28
-T n.m.r. 20 MHz
,amb.temp.
ro -po
100.3? 101.00 101.33
97.14 95/ 97 95 81 :
93.11
acetone 31-0?
046 X
K46,Compound
P
Spectrum N 0 . 2 9
2
H n.m.r.
400 MHz, water n u l l
acetone 2.23
5.19 5.30
4.85 5.06
4.65
4.69
C. speciosa, Compound A
Spectrum No.31
GlcA - — - Man~OH 1 H n.m.r. 8
400 MHz, water n u l l
acetone 2.23
5.30
J
Spectrum No.32
C. speciosa, Compound GlcA - — - Man - — - GlcA - — - Man~OH ,
3
H n.m.r.
a
3
acetone
400 MHz, water n u l l
2.23
HOD
245
APPENDIX I V USES OF
PERACETYLATED
ALDONONITRILES
246
USES OF PERACETYLATED ALDONONITRILES
P e r a c e t y l a t e d a l d o n o n i t r i l e s have been known s i n c e 1 8 9 3 . They were f i r s t used
i n s y n t h e s i s , a ) the Wohl degradation" ' 1
(pentoses a r e obtained from the p e r a c e t y l a t e d h e x o n o n i t r i l e s ) , b) f o r m a t i o n o f 1 - a m i n o - l - d e o x y a l d i t o l s ,etc.The
synthesis of
these d e r i v a t i v e s has been s t u d i e d and the b e s t c o n d i t i o n s observed were the treatment
o f the aldose w i t h hydroxylamine
hy-
d r o c h l o r i d e i n p y r i d i n e and then a c e t y l a t i o n and d e h y d r a t i o n done a t the same time w i t h a c e t i c anhydride
a t h i g h tempera -
ture-^ ( i n the case o f g l u c o s e . i t has been observed temperature
a cyclic derivative i s preferentially
As d e r i v a t i v e s o f a n a l y t i c a l i n t e r e s t , t h e
t h a t a t low formed).
,the t r i m e t h y l -
4 silylated
oximes
and Jones-* used
were f i r s t used
for g.l.c.
separations.Lance
the p e r a c e t y l a t e d a l d o n o n i t r i l e s f o r g . l . c . s e -
p a r a t i o n o f the methyl
ethers o f D - x y l o s e . S e v e r a l s t a t i o n a r y
phases have been employed s i n c e 1 9 7 1 t o improve the s e p a r a t i o n
6—8 of
the PAAN ( p e r a c e t y l a t e d a l d o n o n i t r i l e s ) ~ . The g . l . c . r e t e n t i o n times and g.l.c.-m.s. f o r the a c e t y l 9 6 1 ated a l d o n o n i t r i l e s o f methyl e t h e r s o f mannose and glucose ' 7
have been reported.The
methylated
sugars i n a m e t h y l a t i o n a-
n a l y s i s can now be c o m p l e t e l y c h a r a c t e r i z e d and i d e n t i f i e d by u s i n g g.l.c.-m.s. o f the d e r i v e d a l d o n o n i t r i l e s and a l d i t o l ac e t a t e s . A l l methyl e t h e r s o f mannose can be separated by g . l . c . as a l d o n o n i t r i l e s
( see Table l ) . I n the course o f t h i s i n v e s -
t i g a t i o n , the p e r a c e t y l a t e d a l d o n o n i t r i l e s o f the methyl were a l s o used as means o f a n a l y z i n g the m e t h y l a t i o n
ethers
products
247 TABLE 1 RELATIVE G.L.C. RETENTION-TIMES OF PERACETYLATED ALDONONITRILES OF METHYL ETHERS OF D-GLUCOSE AND D-MANNOSE. M e t h y l ether —
Retention
times(on
5% o f
butanediol succinate) — D-Glucose
D-Mannose
2 , 3 , ^ , 6-tetra-
0.94
1.00
2,4,6
-tri-
1.45
1.59
2,3.6
-tri-
2.00
1.65
3.4,6
-tri-
1.85
1.89
2,3,4
-tri-
2.00
2.03
2,6
-di-
2.28
4,6
-di-
— —
2,3
-di-
3-50
2.55
3,6
-di-
—
2.85
2,4
-di-
2.84
3.16
3,4
-di-
3-58
3.68
-
2,3,4,6-tetra
nonitrile.etc.
:
2.45
5^0-acetyl-2,3,4,6-tetra-0-methyl-D-gluco-
- Relative to 5 - 0 - a c e t y l - 2 , 3 , 4 , 6 - t e t r a - 0 - m e t h y l -
mannononitrile.Data
from r e f e r e n c e s
9) and 1 0 ) .
TABLE 2 RELATIVE G.L.C. RETENTION TIMES OF PERACETYLATED ALDONONITRILES OF METHYL ETHERS OF SUGARS.
M e t h y l ether -
R e l a t i v e r e t e n t i o n times OV-225 3% -
2,3,4,6 2,4,6 2,4,6 2,4,6 2,3,4 2,3,6 3,4,6 4,6 4,6 3,6 2,3 2,3 2
-
- Glc - Glc - Man - Gal - Glc - Glc - Man - Man - Gal - Glc - Glc - Gal — Glc
1.00 °1.39 1.59 1.65 1.85 1. 98 1.98 2.20 2.44 2.59 2.81 2.81 3.02
2,3,4,6 - Glc : 5-0-acetyl-2,3,4,6-tetra-0-methylglucononitrile.
nitrile. -
Programmed a t 1 6 5 ° f o r 4 min,then a t 2°/min t o 220°,
i s o t h e r m a l f o r 32 min. - 6.6 min .
249
e s p e c i a l l y t o separate the 2,3,4-and ses
2,3,6-tri-0-methylgluco-
(see Table 2 ) . Ketoses
can a l s o be c h a r a c t e r i z e d and separated by g . l . c .
and g.l.c.-m.s. as the p e r a c e t y l a t e d o x i m e s . 11
In g e n e r a l , t h e PAAN have been used f o r examining n e u t r a l 12 13 sugars from p o l y s a c c h a r i d e s . g l y c o p r o t e i n s .mucopolysaccharides
13
.and products r e s u l t i n g from Smith d e g r a d a t i o n
14
.Another
use f o r these d e r i v a t i v e s , i s the d e t e r m i n a t i o n o f the degree o f p o l y m e r i z a t i o n o f o l i g o s a c c h a r i d e s and p o l y s a c c h a r i d e s as w e l l as the i d e n t i f i c a t i o n o f the r e d u c i n g end.The g e n e r a l proce dure,known as the M o r r i s o n procedure -*, i n v o l v e s the r e d u c t i o n 1
w i t h NaBH^ o f the r e d u c i n g e n d , h y d r o l y s i s , a n d treatment h y d r o l y z a t e w i t h hydroxylamine lowed by a c e t i c anhydride.The
o f the
hydrochloride i n pyridine f o l f r e e sugars a r e converted
into
the PAAN and the a l d i t o l i n t o the a l d i t o l a c e t a t e . A f t e r g . l . c . s e p a r a t i o n and q u a n t i t a t i o n , i t i s p o s s i b l e t o determine the r a t i o o f f r e e s u g a r / r e d u c i n g end which g i v e s the D.P.By i d e n t i f i c a t i o n o f the a l d i t o l a c e t a t e , t h e r e d u c i n g end i s d e t e r mined. A l d i t o l a c e t a t e s have been used f o r c d . measurements ^, 1
to determine ars
the a b s o l u t e c o n f i g u r a t i o n o f t h e i r parent
sug-
( D or L ).Sugars whose a l d i t o l s are meso compounds
( g a l a c t o s e , x y l o s e , e t c . ) c a n n o t be s t u d i e d , a s they do not show cd.
a c t i v i t y . T h e p e r a c e t y l a t e d a l d o n o n i t r i l e s , a s they keep
the c h i r a l i t y o f the parent sugars and chromophores a r e pres e n t , show c d . a c t i v i t y and can be used t o determine s o l u t e c o n f i g u r a t i o n o f the parent sugars.They
the ab-
can be e a s i l y
250
TABLE 3 CIRCULAR DICHROISM
Peracetylated
OF THE PERACETYLATED ALDONONITRILES.
Configuration
aldononitriles
S i g n o f the c.d. curve
Arabinose
L
-
Arabinose
D
+
Fucose
L
-
Fucose
D
+
Galactose
D
+
Glucose
D
+
Mannose
D
+
251
prepared and s e p a r a t e d by p r e p a r a t i v e g . l . c . R e s u l t s o f t h i s i n v e s t i g a t i o n are g i v e n i n Table 3 « Experimental. P r e p a r a t i o n o f the p e r a c e t y l a t e d The f r e e
aldononitriles.
sugars ( 5 - 1 0 mg) a r e d i s s o l v e d i n hydroxylamine
hydrochloride i n pyridine f o r 1 5 min.The s o l u t i o n
( 5 ^ , 1 mL) and heated on a steam-bath
i s cooled t o room temperature and a c e t i c
anhydride ( 1 mL) i s added and heated f o r 1 h on a steam-bath. Water ( 5 - 1 0 mL) i s added and the PAAN a r e e x t r a c t e d w i t h CHCl^. A f t e r removal o f the s o l v e n t , t h e samples a r e ready f o r g . l . c . S e p a r a t i o n o f the PAAN by g . l . c . The PAAN d e r i v e d from the f r e e sugars were s e p a r a t e d on a column o f 3 % O V - 2 2 5 on Gas Chrom Q and the temperature used was 2 1 0 ° isothermal.The PAAN d e r i v e d from methylated sugars were separated on the same column,with the temperature programme 1 6 5 ° f o r 4 min,then 2 % i i n
t o 2 2 0 ° f o r 3 2 min.
C i r c u l a r d i c h r o i s m measurements o f the PAAN. Samples o f the PAAN i s o l a t e d by p r e p a r a t i v e g . l . c . on O V - 2 2 5 3% ( i s o t h e r m a l 2 1 0 ° ) were d i s s o l v e d i n a c e t o n i t r i l e and the
c d . curves were measured.
252
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