Organic compounds -- part of the class 532-570 series – Organic compounds – Heterocyclic carbon compounds containing a hetero ring...
Patent
1994-09-14
1996-05-28
Springer, David B.
Organic compounds -- part of the class 532-570 series
Organic compounds
Heterocyclic carbon compounds containing a hetero ring...
5483151, C07D49504
Patent
active
055213197
DESCRIPTION:
BRIEF SUMMARY
The present invention concerns a new biotinylation reagent, a process for its production, its use for biotinylating substances with free primary or/and secondary amino groups and a conjugate which is formed by reaction of the biotinylation reagent with substances containing amino groups. In addition the invention concerns a method for the determination of an analyte using the conjugate according to the invention as well as a reagent for the determination of an analyte that contains a conjugate according to the invention.
The binding pair comprising the binding partners biotin and streptavidin/avidin is of very great importance for methods in the field of medicine, biochemistry, molecular biology and immunology. The reason for this is that biotin or a biotin derivative that is bound to another substance is still capable of a high-affinity interaction with streptavidin or avidin and the biotin streptavidin/avidin complex formed in this manner represents an excellent detection system for biological substances.
Biotinylation of various substances such as proteins, peptides, nucleic acids, nucleotides or haptens using a biotinylation reagent has been known for a long time. Nowadays biotin active esters, in particular biotin-N-hydroxysuccinimide esters (biotin-NHS) (E. A. Bayer and M. Wilchek in Methods of Biochemical Analysis, Vol. 26 (Editor: D. Glick), Interscience Publication, J. Wiley & Sons, p. 2-45) and biotin-.epsilon.-amino-caproic acid-N-hydroxysuccinimide esters (biotin-X-NHS) (R. H. Burdon and P. H. von Knippenberg in Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 15 (Editor: P. Tijssen), Elsevier, p. 23-38; S. M. Costello et al., Clin. Chem. 25/9 (1979), p. 1572-1580) are the most common biotinylation reagents. Biotin when in the form of these active esters can be coupled via stable amide bonds to a reaction partner with primary or/and secondary amino functional groups without structurally impairing its heterobicycle moiety that is relevant for its binding capability to avidin/streptavidin. Since especially primary amino functional groups in the form of .epsilon.-amino groups of the lysine side chains occur in almost every protein, the aminolysis of the active esters has been established as the method of choice for biotinylating proteins such as antibodies or enzymatically active proteins and also haptens such as steroid hormones (L. X. Tiefenauer and R. Y. Andres, J. Steroid Biochem. 35 (1990), p. 633-639). Therefore biotin-NHS and biotin-X-NHS are also sold by numerous manufacturers of fine chemicals and bioreagents.
It has also been known for a long time that in biotinylation reagents it has proven to be advantageous to introduce a spacer between the biotin unit and the reaction partner to be biotinylated (N. M. Green et al., Biochem. J. 125 (1971), p. 781-791). Spacers with amphiphilic structures have proven to be particularly useful for this (see e.g. EP 0 410 280 A1; EP 0 451 810 A1).
A disadvantage of the previously known biotin active esters is, however, that they are only sparingly soluble or in some cases even insoluble in almost all common organic solvents. For example biotin-NHS and in particular biotin-X-NHS are only moderately soluble in organic solvents with the exception of DMSO and DMF. The two aforementioned biotinylation reagents also exhibit only moderate solubility in water and aqueous buffer solutions. Although the commercially available biotinylation reagent sulfo-NHS-LC-biotin (I. M. Grumbach and R. W. Veh, J. Immunol. Meth. 140 (1991), p. 205-210) which is a sulfonated variant of biotin-X-NHS exhibits a good solubility in an aqueous medium, it is, however, on the other hand not soluble in organic solvents in the required concentrations. The latter has disadvantageous consequences in particular in the synthesis of hapten-biotin conjugates which is preferably carried out in organic solvents such as dioxane, DMF etc. (L. X. Tiefenauer and R. Y. Andres, J. Steroid Biochem. 35 (1990), p. 633-639; EP 0 451 810 A1).
The European Patent Application EP-A-0 156 287
REFERENCES:
Hofmann et al, Biochemistry, 1984, 23, 2547-2553.
Hoyer et al, J Am. Chem. Soc., 1990, 112 3249-3250.
Hoess Eva
Huber Erasmus
Lenz Helmut
Zink Bruno
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