67
afc
Reactive resins - require activation for ligand
attachment
Ligands may be coupled to
TOYOPEARL AF-Formyl-650M
(aldehyde-bearing) resin under mild conditions exclusively
using primary amines. The ligand is bound to the resin by
a stable secondary amine linkage (Figure 4). Representative
coupling capacities are shown in Table II.
A wide variety of industrial enzymes have been immo-
bilized on aldehyde-bearing supports. Typically, these
supports have been synthesized by industrial users by
partial oxidation of polysaccharide supports (e.g. cellulose
and agarose) or partial hydrolysis of polyacetals. In
contrast, TOYOPEARL AF-Formyl-650M resin is a ready-
to-use aldehyde support formulated from a dimensionally
stable, macroporous matrix. Consistent aldehyde content
and physical properties are assured from batch to batch.
TOYOPEARL AF-Amino-650M
resin may be used to couple
ligandsusing their carboxyl groups (peptidebond formation)
or aldehyde groups (reductive amination) as shown in
Figure 5. Aldehyde groups may be
present in a carbohy-
drate or glycoprotein ligand or may be introduced into the
ligand by mild, periodate oxidation.
The optimized functional group density of TOYOPEARL
AF-Amino-650M (100 mmol/L) is ideal for coupling of either
proteins or low molecular weight ligands. For example,
lactose was coupled by reductive alkylation to yield a
ligand density of ca. 30 µmol/mL resin. Coupling densities
for various proteins are given in Table II.
TOYOPEARL AF-Carboxy-650M
resin provides another
useful and milder approach for coupling to amino groups
of proteins or low molecular weight ligands. The carbodi-
imide mediated coupling reaction produces an amide bond
between ligand and support (Figure 6). Representative
coupling densities are given in Table II.
Affinity
chromatography
figure 6
TOYOPEARL AF-carboxy Coupling Procedure
-O-R-O-CH
2
COOH + HN-Ligand
Toyopearl AF-Carboxy
carbodiimide
pH: 4-6
25 °C
-O-R-O-CH
2
-CO
2
-NH-Ligand
Peptide bond linkage
R = hydrophilic polymer
HW-
65
HW-
65
figure 5
TOYOPEARL AF-amino Coupling Procedure
NaCNBH
3
HW-
65
Schiff’s Base Intermediate
R = hydrophilic
-O-R-O-CH
2
-CHOH-CH
2
-N=CH-Ligand
HW-
65
-O-R-O-CH
2
-CHOH-CH
2
-NH-CH
2
-Ligand
pH: 7
25 °C
-O-R-O-CH
2
-CHOH-CH
2
NH
2
+ HOOC-Ligand
Toyopearl AF-Amino
HW-
65
-O-R-O-CH
2
-CHOH-CH
2
NH-CO-Ligand
Peptide bond linkage
HW-
65
carbodiimide
pH: 4-6
25 °C
-O-R-O-CH
2
-CHOH-CH
2
NH
2
+ OHC-Ligand
HW-
65
Stable secondary amine linkage
Toyopearl AF-Amino
figure 4
TOYOPEARL AF-formyl Coupling Procedure
-O-R-O-CH
2
-CHO + H
2
N-Ligand
Toyopearl AF-Formyl
HW-
65
-O-R-O-CH
2
-CH=N-Ligand
Schiff’s Base Intermediate
(Unstable)
HW-
65
R=hydrophilic polymer
pH: 7
25 °C
NaCNBH
3
-O-R-O-CH
2
-CH
2
-NH-Ligand
HW-
65
Stable secondary amine linkage
