Organic compounds -- part of the class 532-570 series – Organic compounds – Heterocyclic carbon compounds containing a hetero ring...
Patent
1998-08-27
1999-06-15
Richter, Johann
Organic compounds -- part of the class 532-570 series
Organic compounds
Heterocyclic carbon compounds containing a hetero ring...
548547, 548548, 530350, C07D207404, C07D207408, C07D207452, C07K 110
Patent
active
059123591
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
This invention relates to dimaleinimido-substituted compounds, to a process for their preparation, to their use as cross-linking reagents, to the preparation of cross-linked proteins therefrom, to a method for decreasing the molecular weight of proteins thus-cross-linked by means of a cleavage reaction, and to methods, including analytical methods, for lowering the solubility of proteins or for making them more easily removable from an environment or a medium such as a cell or a solution, through cross-linking, and subsequently recovering or restoring the non-cross-linked protein from the cross-linked protein thus removed or insolubilized, so that the restored or recovered protein can be detected or isolated or more accurately characterized.
DESCRIPTION OF THE PRIOR ART
In the realm of protein research, widespread use is made especially of bifunctional cross-linking reagents ("cross-linkers"). These are substances which have two functional groups which are either identical (homobifunctional cross-linking reagents) or else different (heterobifunctional cross-linking reagents). The functional groups react with the corresponding reactive amino acid lateral chains of proteins, thus forming bridges between the side chains. The bridge formation can take place either on the intramolecular level and thus be employed, for instance, to determine distances between two adjacent reactive groups in the protein such as, for example, in the active center of enzymes, or else the bridge formation takes place on the intermolecular level between two or more protein molecules, so that higher molecular complexes are formed. Such intermolecular cross-linking reagents can be employed, for instance, to analyze antigen-antibody interactions, the structure of multi-enzyme complexes, the arrangement of proteins in membranes and many other biological environments. In addition to non-cleavable bifunctional cross-linking reagents, there is also widespread use of cleavable cross-linking reagents.
Proteins (and polypeptide chains in general) can be cross-linked in variety of ways. Some proteins have free sulfhydryl (--SH) groups (also called thiol or mercapto groups) available on the polypeptide chains, and these groups are reactive toward various cross-linking agents. Mercaptalbumin, for example, has a single reactive thiol group on each molecule, hence cross-linking of mercaptalbumin results in the formation of a "dimer". See
Cross-linking reagents which are reactive with respect to SH groups include, among others, bismaleinimido-substituted aliphatic or aromatic compounds such as, for example, the uncleavable compounds dimaleinimidohexane (M. D. Partis et al. (1983), J. Prot. Chem. 2, 263), 4,4'-dimaleinimidostilbene (P. Chantler, S. M. Bower (1988), J. Biol. Chem. 263, 938) and p-phenylenedimaleinimide (J. E. Moore, W. H. Ward (1956), J. Am. Chem. Soc. 78, 2414), maleinimidomethyl-3-maleinimido-propionate that can be cleaved by bases (S. Sato, M. Nakao (1981), J. Biochem. 90, 1177) and several bis(maleinimidoalkoxy)-compounds that can be cleaved with acids (K. Srinivasachar, D. M. Jr. Neville (1989), Biochemistry 28, 2501).
A disadvantage of the cleavable bismaleinimido derivatives used so far, which react with SH groups, is that the subsequent cleavage of the cross-linking reagents from the proteins must take place in acidic or alkaline media; these reaction conditions can cause a denaturation of the proteins, so that it is no longer possible to functionally detect them after the cleavage reaction. Moreover, undesired side reactions often occur during the cleavage reaction or during the acidic or alkaline hydrolysis.
The prior art bismaleinimido compounds can be considered to have the structure of formula IV ##STR2## where R is the bridging radical which joins the heterocyclic rings and is generally either non-cleavable or is cleaved under relatively severe conditions.
According to Moore et al, op. cit., cross-linking of proteins with bis-maleimides can occur via the following mechanism. ##STR3## The greatl
REFERENCES:
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Srinivaschar, K. et al, "New Protein Cross-linking Reagents That Are Cleaved by Mild Acid", Biochem. 28, pp. 2501-2509 (1989).
Feit, P. W. et al, "Alkylating Agents Related to 2,2'-Biaziridine,. I. Compounds Derived from 1,4-Diamino-2,3-butanediol", J. Med Chem. 10, pp. 697-700 (1967).
Moore, J. E., et al, J. Am. Chem. Soc. 78:2414-2418 (1956).
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Bauer Paul J.
Hagen Volker
Forschungsverbund Berlin E.V.
Forschungszentrum Julich GmbH
Keating Dominic
Richter Johann
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