Affinity Chromatography - Department of Molecular and Cellular ...

Coupling through a thiol group
Thiopropyl Sepharose 6B
The active thiol groups of Thiopropyl Sepharose 6B (see page 91 for product details) can be
used to couple many types of small ligands to synthesize affinity media.
• Heavy metal ions and derivatives can be used as ligands to react with thiol groups
forming mercaptides.
• Alkyl or aryl halide ligands give thioether derivatives.
• Ligands containing C=O, N=N and, under certain conditions, C=C bonds undergo
addition reactions.
The medium is converted into the free thiol form, as described earlier, before ligands can be
coupled. The hydroxypropyl group acts as a small spacer arm. Reactions of free thiol
groups are shown in Figure 70.
S
S
Covalent
Chromatography
3
2
DTT
1
PDS
N
S
Hg
COOH
S
S R
1
2
4
5
S
NO 2
SH
5
S
CH2 CONH2
6
S
6
N
R
NHR
6
6
S
O
S
CH
OH
R
S
CH
CH 2
CHO
O
Fig. 70. Reactions of thiol groups. Mixed disulphide formation (1), reversible by reducing agents such as dithiothreitol
(DTT) (2). Mixed disulphide formation with 2, 2'-dipyridyl disulphide gives a 2-thiopyridyl derivative suitable for use
in covalent chromatography (3). Reaction with heavy metals and their derivatives e.g. p-chloromercuribenzoate (4)
leads to mercaptide formation. Treatment with alkyl or aryl halides gives thioether derivatives (5). Addition reactions
(6) are possible with a wide variety of compounds containing C=O, C=C and N=N bonds.
Use Thiopropyl Sepharose 6B in the activated form to couple thiol-containing low molecular
weight ligands, such as coenzyme A. If the ligand:protein interaction is so strong that
elution requires denaturing conditions, the entire ligand-protein complex may be eluted
by reduction with dithiothreitol or 2-mercaptoethanol.
120