Requirements for Structural

Determination of a Carbohydrate
O
O
A O
B
-Identification of sugars. C
-Stereochemistry of each sugar.
-Types of linkages.
-Types of ring structures.
-Anomeric configuration of each sugar.
-Sequence of the different sugar residues.
Preparation of Alditol Acetates

OH
O OH
O + O
HO
HO H
OH HO
OH OH

NaBD
4

CHDOAc

COAc
OH
OH
AcOCH Ac2O/Pyr. HO
HO
COAc CHOH
OH
CHOAc D

CH2OAc
 GLC Profile of Alditol Acetates
(Supelco SP2330 Column)
Rha
Fuc

Ara
Rib

Glc
Xyl
Detector Response

Man

Gal

Hep
Inos.

5 10
Minutes
 Preparation of Trimethylsilyl (TMS)
Methyl Glycosides
O O
O
( C OH) ( C OCH3 )
OH ( C OCH3 )
O OH
O + O OH
HO O
HO H HO
HO OCH3 +
OH CH3OH HO
OH
OH
+ OCH3

(CH3)3SiX
O
O
( C OCH3 )
( C OCH3 )
OTMS
O OTMS
TMSO O
TMSO OCH3 +
TMSO + furanose forms
OTMS TMSO
OTMS
OCH3
 Detector Response

Ara
Rha
Fuc
Xyl

GlcA

10
GalA

Man

Gal
GalA
Gal
Glc
GlcA
Glc

Minutes
Hep
Hep

20 Inos.
(J&W Scientific DB1 column)
GLC of TMS Methyl Glycosides
Determination of the Stereochemical
Configuration of Glycosyl Residues
CH2 OTMS CH2 OTMS

O O
OTMS OMe MeO TMSO

OTMS OTMS
OTMS OTMS
2,3,4,6-tetra-O-trimethylsilyl- 2,3,4,6-tetra-O-trimethylsilyl-
methyl -D-glucoside methyl -L-glucoside
Enantiomers: Identical physical properties, unable to separate.

CH2 OTMS CH3 CH2 OTMS

O CH3 CH2 O
OTMS O O TMSO

CH2 CH3 OTMS

OTMS
OTMS CH3 OTMS

2,3,4,6-tetra-O-trimethylsilyl- 2,3,4,6-tetra-O-trimethylsilyl-
2-(-)-butyl -D-glucoside 2-(-)-butyl -L-glucoside
Diastereomers: Different physical properties, able to separate.
 GC Separation of Trimethylsilyl
(-)-2-Butyl Glycosides

L+D-Glc
D-Man
L-Man
D-Fuc

D-Fuc

D-Gal

D-Gal
L-Fuc
L-Fuc

D-Glc
L-Gal

L-Gal
L-Gal
D-Xyl

D-Xyl

L-Glc
L-Xyl

L-Xyl
STD
Detector Response

10 20 30 40
Minutes
Gerwig, G.J., Kamerling,J.P., and Vliegenthart, J.F.G. 1978. Carbohydr. Res. 62:349-357
 Glycosyl Linkages by Preparing Partially
Methylated Alditol Acetates (PMAAs)
O

CH3 SCH3
OH
HO
O KH
HO OCH3
HO O
OH O H3 CO
HO H3 CO
O OH + - H3 CO
K CH2 SCH3 CH3 I
O OCH3 H3 CO
OH
OHO OH OCH3
O O
HO H3 CO OCH3
OCH3 O
HO O O O O
H3 CO
OH
OR H3 CO O O
OCH3
- OR
B - R2I
R1OH R1O R1OR2
Ether synthesis

2 M TFA

CHDOAc CHDOAc
CHDOAc CHDOAc
OMe OAc
MeO OMe NaBD 4
MeO MeO Ac2O/Pyr
MeO + AcO +
+
OMe OMe
OMe AcO
OAc OAc
OAc OAc
CH2 OMe CH2 OMe
CH2 OMe CH2 OMe
Primary Fragments for PMAAs
R R
R
.+ +
H C O Me H C. OMe
H OMe
+ = +
H OMe .
H C OMe H C
+
O Me
R'
R' R'

R R
R
.+ H C
+
O Me H C. OMe
H OMe
+ >> +
H OAc .
H C OAc +
R' H C O Ac
R' R'

R R
R
.+ +
H C O Ac H C. OAc
H OAc +
H OAc = +
.
H C OAc +
R' H C O Ac
R' R'
Primary and Secondary Fragments of
the PMAA of Terminal Glucose

CHDOAc

OMe 118
MeO 205
162
161 OMe

OAc 45
CH2 OMe
Primary Fragments

Secondary Fragments
+ +
CH O Me CH O Me
H C OMe - HOAc H C OMe
H C OAc H C
CH2 OMe CH2 OMe
m/z 205 m/z 145

CHDOAc CHD
- HOAc
H C OMe H C OMe
+
H C O Me +
H C O Me
m/z 162 m/z 102
+ +
CH O Me CH O Me
- HOAc
H C OAc H C
CH2 OMe CH2 OMe
m/z 161 m/z 101

-MeOH
+
CH O Me
+
O CH O Me
H C C O - CH2CO
C O
CH2 CH2
CH3
H

m/z 129 m/z 87

 Reduction with Borodeuteride and
Determination of Glycosyl Ring Size
CHD(H)OAc CHD(H)OAc
OAc 305 OMe 118(117)
MeO 233 AcO
190(189) 189
OAc OMe 234(233)
306(305) OMe 117 OAc
CH2OAc CH2OAc
 Detector Response

5
t-linked-Araf

10
t-linked-Arap

2-linked-Araf
3-linked-Araf

15
4-linked-Arap
2-linked-Arap
3-linked-Arap

Minutes
2,3-linked-Araf

20
3,4-linked-Arap or 3,5-linked-Araf
2,3-linked-Arap
2,5-linked-Araf and 2,4-linked Arap
GLC Profile of Arabinosyl PMAAs

2,3,4-linked-Arap and 2,3,5-linked-Araf

25
 Glycosyl Linkage Determination of a
Uronosyl Residue
O OCH3
O C
HOOC
O O + -
K CH2 SCH3 CH3 I O
O
HO O H3CO O
OH OCH3
CDHOAc

OMe 118
MeO
233
OAc
OAc LiB(CH2 CH3 ) 3 D
CH2 OMe
PMAA of a 4-linked
CHDOAc Glc residue

118 OMe
CD2OH
+
263 MeO Ac2 O NaBD4 H O O
Pyr. O
OAc H3CO
OCH3
OAc
CD2OAc
Glycosyl Linkage Determination of an
Acid-Labile Residue (Kdo)
O CH2 OH
HO
O
O O CH2 OMe
HO COOH MeO
+ - O O
K CH2 SCH3 CH3 I
O MeO C
OMe
O

LiB(CH2 CH3 ) 3 D

O CH2 OMe
MeO
O

MeO CD2 OH

O
+
Mild H
CD2 OAc

D OAc NaBD4
CH2

MeO
O CH2 OMe
AcO + MeO
Ac2 O NaBD4 H OH
OAc Pyr. CD2 OH
MeO
OMe D
OH
CH2 OMe
Linkage Determination of an Oligosaccharide
Reducing Glycosyl Residue

OH
O OH
O
O OH D
HO OH NaBD 4
OH HO OH
OH
H
H
CDHOAc
OMe 118
MeO

OAc
233 CDHOM e 1. Methylation
2. Hydrolysis
OAc
CH2 OMe
90 OM e 3. Reduction
PMAA of a 4-linked
Glc residue 4. Acetylation
205 MeO

OA c
OM e 250
CH 2 OM e 45
Determining a Carbohydrate Sequence

Glycoconjugate Enzymatic Oligosaccharides

-Glycosyl Compositon Chemical -Separation
-Glycosyl Linkages -Glycosyl Compositon
-NMR Spectroscopy -Glycosyl Linkages
-NMR Spectroscopy
-Mass Spectrometry

Sequence of
Oligosaccharides
Elimination of Uronosyl Residues
PS OMe MeO O OMe
O
O C OMe
O O
MeO O
OMe PS
MeO O
OMe OMe

Base

O
OS OMe MeO OMe
O C
O OMe
O O
MeO OH
OMe OS
MeO O
OMe OMe

Base or
Mild Acid

OMe
OS OMe OMe
O O
O
MeO OH OS
HO
OMe OMe

1. Reduction
2. Ethylation (dimsyl ion followed by ethyl iodide).

OMe
OS OMe OMe
O
O OEt D OS
MeO OEt EtO
OMe OMe
H
Lithium Degradation of Uronosyl Residues

PS OH HO O OH
O
O C OH
O O
HO O
OH PS
HO O
OH OH

1. Lithium degradation (ethylene diamine + Li)

2. Reduction

OH OH
OS
O OH D OH
O
HO OH OS
OH HO
H OH
 Degradation of Phosphorylated
Polysaccharides
-
OPO3
PS OH OH
O O O OPO3 Me2
O
PS PS OMe OMe
HO O P O O O
O O
OH OH
- PS
O MeO O O
P
OMe OMe
OMe
48% Aqueous HF at 4 degrees.
48% Aqueous HF at 4 degrees.
OH
PS OH OH
O O OH
O
OH HO PS PS OMe OMe
HO O O
OH OH O
OH HO PS
MeO
OMe OMe

A. 1. Reduction
2. Ethylation

OEt
PS OMe OMe
OEt D O
O
OEt EtO PS
MeO
OMe OMe
H

B.
 Smith Degradation
OH
OH
O
OH OH
O O
O O HO
PS OH HO O PS
OH OH

NaIO4

OH HO
OH
O
OH
O O O
O HO
OH C PS
PS O
O H C OH
H O

NaBD4

Mild H+

OH CH2OH
OH OH OH
O O
HO
O
O OS
HO
OH CH2OH OH
OS