Organic compounds -- part of the class 532-570 series – Organic compounds – Heterocyclic carbon compounds containing a hetero ring...
Reexamination Certificate
1996-10-18
2001-04-17
Higel, Floyd D. (Department: 1626)
Organic compounds -- part of the class 532-570 series
Organic compounds
Heterocyclic carbon compounds containing a hetero ring...
C435S007100, C435S007400
Reexamination Certificate
active
06218546
ABSTRACT:
DESCRIPTION
The invention concerns compounds which have a chromophore and a group capable of binding to streptavidin or/and avidin and are suitable for binding to molecules which contain an aldehyde, ketone, semiacetal or/and semiketal group. In addition the invention concerns conjugates formed from these compounds as well as a method for the detection or isolation of carbohydrates or glycan receptors by means of such conjugates.
Glycoconjugates are found in many biological areas for example as enzymes, transport proteins, receptor proteins, hormones or structural proteins. The interactions of free saccharides or saccharide moeities of the glycoconjugates with specific receptors play an important role in the biological function of these compounds.
In order to be able to investigate these interactions the appropriate saccharides have to be obtained in as pure a form as possible. Due to the great diversity of saccharides in natural samples and the structural similarity of many saccharides, it is, however, difficult to isolate pure defined saccharides. In particular the low UV sensitivity of saccharides hinders the detection of small amounts.
Various methods are known which are used to fractionate and detect saccharides. Analysis by means of high pH anion exchange (HPAE) chromatography with pulsed amperometric detection (PAD) allows the direct quantification of saccharides with a simple sample preparation without requiring a derivatization. However, the disadvantage of this method is that the strong basic conditions on the one hand have a detrimental effect on the saccharides and on the other hand they cannot be used directly in binding studies.
In a further method the saccharides are firstly derivatized. For this the saccharides are labelled with UV or fluorescence-active reagents such as e.g. by derivatizing with 2-aminopyridine (Rice et al., Anal. Biochem. 206 (1992), 278-287). The disadvantages of the method described in this reference are the high reaction temperatures which can lead to a decomposition of the saccharides (Kakehi et al., Anal. Biochem. 199 (1991), 256) and the use of toxic reagents such as e.g. NaCNBH
3
. Moreover the conjugates obtained cannot be used directly in binding studies.
Methods for characterizing glycan-receptor interactions have also been described. In these methods soluble saccharides are derivatized in such a way that they can be bound to a solid phase or to macromolecules. This enables an examination of interactions between receptor and saccharide. For example in the method described by Feizi et al. (Feizi et al., Methods Enzymol. 230 (1994), 484-519) the saccharides are derivatized with a lipid and separated on thin layer plates. Subsequently the binding studies are carried out directly on the plate. However, a disadvantage of this method is the low sensitivity of the staining and detection of the saccharide. Moreover the separation is limited to a single separation technique namely thin layer chromatography. In addition the derivatization can be achieved by coupling oligosaccharides to neoglyco-proteins (Hubert et al., Cell. Diff. Dev. 27 (1989), 69) or directly to biotin. The derivatives obtained in this manner are, however, unsuitable for a conventional fractionation and detection so that firstly other methods have to be found for their purification.
Toomre and Varki prepared and used the reagent 2-amino6-amidobiotinyl-pyridine to attempt the detection and fractionation of saccharides and their direct characterization in binding studies with a single reagent (Toomre et al., Glycobiol. 4 (1994), 653-663). In this process a conjugate is formed from the saccharide and the reagent containing an amine group. Apart from the amine group the reagent also has the fluorescent properties of 2-aminopyridine and the affinity of biotin to streptavidin. As a result it was possible to carry out binding studies directly after a sensitive separation. However, disadvantages of this method are the different coupling efficiencies depending on the saccharide which are referred to in the patent application WO94/28008 by Varki and Hotenberg, the possible decomposition of the saccharides under the reaction conditions and the toxicity of the reducing agent.
Moreover as a result of the reductive amination step the sugar is always in an open-chain form at the reducing end and is no longer able to form a closed ring structure. Thus the formation of the conjugate modifies the structure of the saccharide. Since, however, it is the saccharide which represents the recognition structure which is to be examined, the specific receptor for the saccharide may possibly not bind to the saccharide which is a major disadvantage of this method.
Detection reagents that are suitable for examining the interactions of saccharides with receptors should ideally have the following properties:
the test reagent should contain a marker group by means of which the saccharides can be detected with high sensitivity by UV/VIS or fluorescence spectrometry,
the conjugate which forms by coupling the saccharide to the detection reagent should be capable of binding to macromolecules or solid phases: this property can be used either to immobilize or to detect the saccharide,
the saccharides should not be decomposed under the reaction conditions,
it should be possible to have a closed ring structure at the reducing end of the sugar in the derivatization,
it should be possible to separate derivatized saccharide mixtures in a simple manner,
it should be possible in a relatively simple manner to separate the detection reagent which is usually used in excess for the derivatization from the reacted saccharides,
the reagents required for the derivatization should have the lowest possible toxicity or be non-toxic,
the efficiency of the coupling reaction should be high for all saccharides,
the detection reagent should facilitate an analysis by means of mass spectrometry,
the detection reagent should not influence the interaction between the saccharide and the ligand.
One object of the present invention was therefore to provide improved reagents for the detection and isolation of saccharides and glycan receptors.
This object is achieved according to the invention by a compound having a structure of the formula (I):
x—y (I)
in which X represents a residue selected from —NR—NH
2
, —C(A)—NR—NH
2
or —B—C(A)—NR—NH
2
in which R is hydrogen or an alkyl residue with 1 to 4 C atoms and A and B each independently represent NH, O or S and Y represents a residue which contains a chromophore as well as a ligand capable of binding to streptavidin or/and avidin.
Such a compound can be coupled via the residue X to ketone, aldehyde, semiketal or semiacetal groups which in particular are present in saccharides. The resulting conjugates have a chromophore group and are capable of binding to streptavidin or avidin. The compound (I) preferably has a high absorption value &egr; in the UV/VIS range and thus enables a sensitive detection of the substance to be detected. In addition the substance to be examined can be coupled to a solid phase or a macromolecule by the ligand capable of binding to avidin or/and streptavidin. As elucidated in more detail in the following the compound (I) can be coupled to saccharides under reaction conditions in which the saccharides are not decomposed. Furthermore no toxic reagents are required for the reaction. The compound (I) reacts selectively with the reducing end of saccharides while retaining their ability to form a closed ring structure. Moreover detection reagent added in excess can be simply separated by means of phase separation of the conjugates obtained.
X can represent a residue —NR—NH
2
which is bound to an alkylene, alkenylene or alkinylene group or to an aromatic group such as a hydrazine. In addition X can represent the group —B—C(A)—NR—NH
2
in which A, B and R are defined as above such as a semicarbazide, or a group —C(A)—NR—NH
2
such as a hydrazide.
It is preferred according to the invention that the chromophore which is contained in the residue Y has an absorption ma
Fernholz Erhard
von der Eltz Herbert
Watzele Manfred
Arent Fox Kintner Plotkin & Kahn
Higel Floyd D.
Roche Diagnostics GmbH
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