Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – Chemical aftertreatment – e.g. – acylation – methylation – etc.
Reexamination Certificate
2000-01-18
2001-06-26
Brusca, John S. (Department: 1631)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
Chemical aftertreatment, e.g., acylation, methylation, etc.
C544S369000, C544S373000, C548S236000, C548S511000
Reexamination Certificate
active
06252042
ABSTRACT:
The present invention relates to complexes (markers) especially for the detection of phosphoric esters, the complexes comprising a chelating agent, a central atom and a fluorescent dye.
Phosphoric esters are vital components of biological systems and are constituents of a large number of biochemical compounds. They play an important role in particular in the functioning and the regulation of cellular processes, occurring, for example, as intermediates in a large number of metabolic processes. Biologically important phosphoric esters include, for example, adenosine mono-, di- and tri-phosphates, lecithins, cephalins, phospholipids, nucleotides and co-enzymes. An especially large part in regulatory processes is also played by the phosphorylation and dephosphorylation of proteins by proteinkinases and phosphatases, respectively. The detection of phosphorylated molecules, that is to say the analysis of the phosphorylation state of biomolecules, is accordingly of considerable importance in biological research.
The radioactive labelling of phosphorylated compounds with
32
P or
33
P and the detection of corresponding radionuclides is already known, but such procedures have the disadvantage that they require extensive safety precautions.
It is also known to use specific antibodies to detect phosphorylated compounds. The preparation of antibodies is complex, however, and therefore expensive, and their specificity is strongly influenced by structures other than the phosphate esters to be analysed. There therefore continues to be a need for alternative compounds for the detection of phosphoric esters.
The object of the present invention is accordingly to provide complexes that can be used for a very sensitive, non-hazardous and specific assay, such as, for example, the detection of phosphoric esters, or for the separation of compounds, such as proteins or peptides, without the need to use radionuclides or antibodies.
The object is achieved by a complex that comprises
a) at least one chelating agent,
b) at least one central atom or central ion coordinated by the chelating agent(s), and
c) at least one dye bonded to the chelating agent(s).
Preference is given to the use of multidentate chelating agents, such as iminodiacetic acid (IDA) and derivatives thereof, nitrilotriacetic acid and derivatives thereof, polyoxycarboxylic acids and derivatives thereof, polyamines and derivatives thereof, and ethylenediamineacetic acid and derivatives thereof.
The selective binding of IDA to phosphate esters by way of Fe
3+
-chelation is already known, see Anderegg, G. & Schwarzenbach, G. (1955) Helv. Chim. Acta 38, 1940-1942; Muszynska, G. et al. (1992) J. Chromat. 604, 19-28; Schwarzenbach, G. et al. (1955) Helv. Chim. Acta 38, 1147-1170; Songyang, Z., Blechner, S., Hoagland, N., Hoekstra, M. F., Piwnica-Worms, H. & Cantley, L. C. (1994), Current Biology 4, 973-981. Also known is the use of immobilised IDA for the affinity purification or concentration of biomolecules on chromatography supports and membranes: for example, such a chelating chromatography material is marketed by the company Pharmacia Biotech under the name Chelating Superose/Sepharose.
A protein purification system is furthermore marketed by the company QIAGEN. That system can be used to isolate proteins and peptides that have been labelled with six consecutive histidine residues. Such residues have a high affinity for immobilised nickel ions, which in turn have been bound to nitrilotriacetic acid resins (Ni-NTA) by chelation. A considerable disadvantage of such a system, however, is the need to label the proteins and peptides with the histidine residues.
Also marketed by QIAGEN are conjugates of Ni-NTA with antibodies, peroxidase, alkaline phosphatase or horseradish peroxidase.
The use of the chelating effect in assays, however, especially for the direct detection and/or the separation of phosphate esters by means of a complex of a chelating agent with a dye (chromophore), is not known.
REFERENCES:
patent: WO9730074 (1997-08-01), None
Yuan et al., “Synthesis of a new tetradentate beta-diketonate-europium chelate that can be covalently bound to proteins in time-resolved fluorometry,” Anal. Sci., 1996, vol. 12, No. 5, pp. 695-699.
Gast Rainer
Gloekler Joern
Tegge Werner
Brusca John S.
Gesellschaft fuer Biotechnologische Forschung mbH (GBF)
Kim Young
Pitney Hardin Kipp & Szuch LLP
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