Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Chemical modification or the reaction product thereof – e.g.,...
Reexamination Certificate
1999-11-22
2001-06-26
Naff, David M. (Department: 1651)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Chemical modification or the reaction product thereof, e.g.,...
C435S176000, C435S177000, C435S178000, C435S179000, C435S180000, C435S188000, C436S524000, C436S528000, C436S529000, C436S530000, C436S531000, C530S811000, C530S812000, C530S813000, C530S814000, C530S815000
Reexamination Certificate
active
06252054
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a method for immobilizing an SH (sulfhydryl) group-containing compound on a solvent-insoluble support.
BACKGROUND ART
As a method for immobilizing a compound on a solvent-insoluble support for use as a ligand for specific adsorption, many investigations have been undertaken in the diverse field of immobilized enzymes, affinity chromatography, ion exchange chromatography, etc. and some methods are used on the industrial application.
As a representative method for immobilization known in the field of immobilized enzymes, there can be mentioned: (1) the method which comprises forming the imidocarbonate on the support by the cyanogen bromide activating method and, then, causing it to react with the amino group of the compound to serve as a ligand; (2) the so-called acid azide derivative method which comprises, sequentially, esterification of the carboxyl group on a support, hydrazide formation, conversion to the azide and substitution thereof with the amino group of the compound to serve as a ligand; (3) the so-called diazo method which comprises forming a diazonium salt on a support and letting it react with the amino group of the compound to serve as a ligand; (4) the so-called condensation reagent method which comprises condensing the amino or carboxyl group on a support with the carboxyl or amino group of the compound to serve as a ligand in the presence of a condensing agent; (5) the so-called alkylation method which comprises modifying a support with bromoacetyl or 4,6-dichloro-s-triazinyl and causing the resulting derivative to react with the amino group of the compound to serve as a ligand; and (6) the so-called matrix-crosslinking method which comprises crosslinking the amino group on a support and the amino group of the compound to serve as a ligand with glutaraldehyde followed by reduction (Atsuo Tanaka & Takuo Kawamoto: Modern Chemistry, 24 to 30, July 1992).
However, those methods have the drawbacks, namely
(a) when the compound to serve as a ligand contains an SH group, the efficiency of immobilization is relatively low; and
(b) when the compound to serve as a ligand is an SH group-containing peptide or protein, in particular, the inherent activity of the compound (enzyme activity, binding activity, etc.) is sometimes sacrificed.
The inventors of the present invention discovered that when a peptide or protein to serve as a ligand contains an SH group, the above-mentioned phenomena (a) and (b) occur during its immobilization on a solvent-insoluble support. Similar phenomena are also observed with compounds other than peptides and proteins provided that they contain SH groups. There is no information in the available literature to the effect that the above phenomena occur when a compound to serve as a ligand is immobilized on an inert support.
SUMMARY OF THE INVENTION
In the above state of the art, the present invention has its objects to provide a method for immobilization which is capable of improving the efficiency of immobilization in the reaction between an SH group-containing compound and a solvent-insoluble support as well as suppressing the deterioration of the inherent characteristic of the SH group-containing compound due to immobilization.
For accomplishing the above object, the inventors of the present invention did an extensive investigation concerning various conditions of the reaction and endeavored to establish a method for immobilization which is capable of improving the efficiency of immobilization and suppressing the deterioration of the inherent characteristics of SH group-containing compounds. As a result, the inventors discovered the involvement of SH group, as a major factor, in the above-mentioned phenomena (a) and (b). Moreover, in view of the fact that those phenomena are liable to occur in reactions conducted in aqueous solution in the high pH (>7) region, the inventors assumed that, under the conditions of immobilization reactions, SH groups are oxidized, for instance, to give the S—S bond.
The S—S bond may at times be formed within the molecule of the compound to serve as a ligand or may also occur between the molecules. It can be thought that when the reaction site (functional group) necessary to be utilized for immobilization of the compound to serve as a ligand is SH group, the formation of the S—S bond inactivates the reaction site and that, when the reaction site is a functional group other than SH group, the reaction site is hidden in the interior of the molecule as the S—S bond is formed. It can be thought that the decrease in immobilization efficiency occurs probably in such situations. Moreover, it can also be assumed that the formation of the S—S bond causes the deterioration of the inherent characteristic of the SH group-containing compound.
Based on the above experience and assumption, the inventors paid attention to the importance of avoiding formation of the S—S bond and, after many investigations, found that the above-mentioned object can be accomplished by conducting the reaction between a solvent-insoluble support and an SH group-containing compound in the presence of an antioxidant. The present invention has been developed on the basis of the above finding.
The present invention, therefore, is directed to a method for immobilizing an SH group-containing compound which comprises reacting a solvent-insoluble support with the above-mentioned compound in the presence of an antioxidant.
REFERENCES:
patent: 4247642 (1981-01-01), Hirohara et al.
patent: 5108916 (1992-04-01), Cobbs et al.
patent: 5298406 (1994-03-01), Loyd et al.
patent: 5824522 (1998-10-01), Ikenaka et al.
patent: 5866387 (1999-02-01), Ogiho et al.
patent: 5874569 (1999-02-01), Elsner et al.
Tanaka et al, Modern Chemistry, Jul. 1992, pp. 24-30.
Nishimoto Takehiro
Nomura Michio
Ogino Eiji
Connolly Bove Lodge & Hutz LLP.
Kaneka Corporation
Naff David M.
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