esters of dicarboxylic acids in forms of p-nitrophenol [11, 12] or Nhydroxysuccinimide (NHS) [13, 14], such as disuccinimidyl glutarate (DSG) [15, 16], disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl) suberate (BS3), and di-(N-succinimidyl) carbonate
(DSC).
In this chapter, we are focused on the conjugation of synthetic
oligosaccharides to carrier proteins through dicarboxylic acid linkers by using activated disuccinimidyl esters of dicarboxylic acids
(Scheme 1) because these linkers have been proven to be efficient
and cause less side effects [17]. Typically, an azido group, which is
stable to most reactions involved in carbohydrate synthesis, is
introduced to the reducing end of free oligosaccharides during
their synthesis as the azido group can be readily converted into a
free amino group to facilitate oligosaccharide conjugation to carrier
proteins. Thus, a detailed procedure for this conjugation method
starting with the reduction of the azido group in synthetic oligosaccharides [16, 18] is provided here (Scheme 1). The remaining
steps of this procedure include condensation of the amino derivative of an oligosaccharide with the dicarboxylic acid linker using a
bifunctional disuccinimidyl ester [15, 19], conjugation of the resultant mono-activated ester of the oligosaccharide antigen to a carrier
protein, and finally analysis of the carbohydrate loading of the
oligosaccharide–protein conjugate [14–16, 19–21].
2 Materials
2.1 Reduction
of the Azido Group
in Oligosaccharide
Antigen
1. Dram vial.
2. Magnetic stir bar.
3. Spatula.
4. Stir plate.
5. Microliter syringe.
6. Rotary evaporator.
2.2 Coupling
of Amino Derivative
of Oligosaccharide
Antigen
with Disuccinimidyl
Ester of Dicarboxylic
Acid Linker (See Note
1)
1. Commercial 0.1 M phosphate-buffered saline (PBS).
2. Dram vial.
3. Magnetic stir bar.
4. Spatula.
5. Stir plate.
6. Syringes (1.0 mL).
7. Buchner funnel.
8. High vacuum pump.
306
Brittany R. Smith and Zhongwu Guo
(DSC).
In this chapter, we are focused on the conjugation of synthetic
oligosaccharides to carrier proteins through dicarboxylic acid linkers by using activated disuccinimidyl esters of dicarboxylic acids
(Scheme 1) because these linkers have been proven to be efficient
and cause less side effects [17]. Typically, an azido group, which is
stable to most reactions involved in carbohydrate synthesis, is
introduced to the reducing end of free oligosaccharides during
their synthesis as the azido group can be readily converted into a
free amino group to facilitate oligosaccharide conjugation to carrier
proteins. Thus, a detailed procedure for this conjugation method
starting with the reduction of the azido group in synthetic oligosaccharides [16, 18] is provided here (Scheme 1). The remaining
steps of this procedure include condensation of the amino derivative of an oligosaccharide with the dicarboxylic acid linker using a
bifunctional disuccinimidyl ester [15, 19], conjugation of the resultant mono-activated ester of the oligosaccharide antigen to a carrier
protein, and finally analysis of the carbohydrate loading of the
oligosaccharide–protein conjugate [14–16, 19–21].
2 Materials
2.1 Reduction
of the Azido Group
in Oligosaccharide
Antigen
1. Dram vial.
2. Magnetic stir bar.
3. Spatula.
4. Stir plate.
5. Microliter syringe.
6. Rotary evaporator.
2.2 Coupling
of Amino Derivative
of Oligosaccharide
Antigen
with Disuccinimidyl
Ester of Dicarboxylic
Acid Linker (See Note
1)
1. Commercial 0.1 M phosphate-buffered saline (PBS).
2. Dram vial.
3. Magnetic stir bar.
4. Spatula.
5. Stir plate.
6. Syringes (1.0 mL).
7. Buchner funnel.
8. High vacuum pump.
306
Brittany R. Smith and Zhongwu Guo