Porphyrin compounds, their conjugates and assay methods...

Details Porphyrin compounds, their conjugates and assay methods... Porphyrin compounds, their conjugates and assay methods...

2001-04-20

2003-06-24

Ceperley, Mary E. (Department: 1641)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving fixed or stabilized, nonliving microorganism,...

C424S009610, C435S006120, C435S040510, C435S040520, C436S546000, C436S800000, C530S391300, C530S391500, C530S402000, C556S136000, C987S003000

Reexamination Certificate

active

06582930

ABSTRACT:

This application is a U.S. national stage of International Application PCT/FI99/00898, filed Oct. 27, 1999 and published on May 18, 2000 in the English language.
FIELD OF THE INVENTION
This invention relates to the chemistry and applications of the metalloporphyrin. In particular it is related to novel photoluminescent metalloporphyrin compounds, their preparation and to the use of the said compounds as labelling reagents for biologically active molecules. This invention can be applied for example, in biomedical research and in-vitro diagnostics, in food industry, in chemical and pharmaceutical industry, in biotechnology as well as in environmental monitoring.
BACKGROUND OF THE INVENTION
Many (bio)analytical systems utilise certain chemical compounds and species as sensitive and specific probes for the determination of specific analytes and parameters. In particular, photoluminescent dyes are frequently used for labelling of biomolecules in different analytical applications. Various photoluminescent dyes with different physical, chemical and spectral properties have been described, that suit a wide range of applications, such as staining of proteins, nucleic acids, cells, for use in microscopy, immunoassays and DNA hybridisation assays. The most common families of the dyes include fluoresceins, rhodamines, coumarines, cyanines and BODIPY dyes (=boron dipyrrylmethine dyes). Families of the dyes which enable high detection sensitivity include photoluminescent lanthanide chelates and phosphorescent metalloporphyrins. A large number of photoluminescent fluorescent dyes and their biomolecule conjugates, such as protein conjugates and nucleic acid conjugates, have been developed in recent years. Many photoluminescent dyes as well as the corresponding labelling reagents and biomolecule conjugates are available commercially (see for example R. P. Haugland. Handbook of fluorescent probes and research chemicals, 6
th
Edn., Molecular Probes Inc., 1996) and widely used for diagnostics and research purposes. Photoluminescent conjugates of biomolecules are usually prepared by contacting a native biomolecule or a derivative of the biomolecule with a reactive derivative of a photoluminescent dye, also called labelling reagent. Examples of reactive groups in common labelling reagents include reactive esters (for example succinimidyl ester, sulphosuccinimidyl esters), isothiocyanato, chlorosulphonato, dichlorotriazinyl and maleimidyl groups. They enable simple and selective labelling of biomolecules via certain functional groups, most commonly via primary amino, thiol or hydroxy groups. Labelling can be performed under mild conditions and without using additional reagents. The availability of such standard labelling reagents and the corresponding photoluminescent bioconjugates is a requirement for development of analytical and diagnostic methods and applications. In many cases it is desirable to covalently attach the dye molecule via a spacer arm (usually 2-20 atoms long), in order to minimise the photochemical and biochemical interactions between the label and the biomolecule.
For those applications where high detection sensitivity is required, such as immunoassays and nucleic acid hybridisation assays, probes with long Stokes' shift and well resolvable spectral characteristics are needed. Significant improvement of sensitivity, especially when working with complex biological samples, can be achieved by using long decay time photoluminescent dyes in combination with time-resolved photoluminescent detection. The time-resolved detection concept allows significant reduction of background interference from scattering and intrinsic fluorescence and consequently provides high signal-to-noise ratio (E. Soini et al. U.S. Pat. No 4,374,120, 1983).
Long decay time photoluminescent lanthanide chelates, in particular the complexes of Eu, Th, Sm, Dy have been suggested for use in sensitive time-resolved fluoroimmunoassays (for example U.S. Pat. No. 4,565,790, U.S. Pat. No. 5,346,996, U.S. Pat. No. 5,571,897). They are presently used in a commercial time-resolved fluoroimmunoassay system DELFIA® (trademark of EG&G-Wallac, Turku, Finland). p-isothiocyanatobenzyl-diethylenetriamine-N
1
,N
2
,N
3
,N
4
-tetraacetic acid chelated with Eu
3+
is now commercially available for labelling of biomolecules, such as proteins, antibodies and nucleic acids.
Fluorescent complexes of ruthenium(II), osmium (II), and some other metals were suggested recently for use as long decay time fluorescent labels, particularly for oxygen sensors and for sensitive fluorescence immunoassays (J. Lakowicz et al., U.S. Pat. No. 5,660,991). Succinimidyl derivatives of Ruthenium bis(2,2′-bipyridine) (2,2′bipyridine-4,4′-dicarboxylic acid) have been described as labelling reagents for electrochemiluminescence immunoassays (U.S. Pat. No. 5,310,687).
A number of photoluminescent porphyrin dyes have been suggested for labelling of biomolecules, particularly for immunoassays (Schmidt D. et al. U.S. Pat. No. 4,614,723; Hendrix J., U.S. Pat. No. 4,707,454; Hendrix J., U.S. Pat. No. 5,464,741; Savitskii A. P. et al., Doklady Akademii Nauk SSSR, 1987, vol. 293, p.744, Braman, J. WO 96/11937). These dyes exhibit intense absorption bands around 400 nm and moderate absorption bands between 500 and 600 nm. Free base porphyrins usually emit fluorescence in red range of visible spectrum, whereas their metal complexes, mainly platinum(II) and palladium(II) porphyrins, exhibit bright phosphorescence in the same wavelength range even at room temperature (D. Dolphin, The Porphyrins, 1978, New York, Academic Press, vol. 3). Such properties make the porphyrin dyes promising in fluorescent and phosphorescent probing of biomolecules and particularly in time-resolved phosphorescence bioaffinity assays. A number of porphyrins as well as certain related structures, mainly biogenic polycarboxy porphyrins and water-soluble derivatives of tetraphenylporphyrin, have been suggested for such applications.
Water-soluble coproporphyrin-I was suggested by Savitsky et al. (Doklady Akademii Nauk SSSR, 1987, vol. 293, p.744) as a label for fluoroimmunoassays. A palladium(II) complex of coproporphyrin-I has been used for phosphorescence labelling of antibodies (Doklady Akademii Nauk SSSR 1989, vol. 304, p. 1005). In similar way some other polycarboxy porphyrins, including coproporphyrin-III, zinc(II)-coproporphyrin-I, dimethoxydeuteroporphyrin IX, hematoporphyrin IX, platinum(II)-coproporphyrin I and III, have been used for labelling various proteins. With these dyes covalent binding to proteins was usually achieved by carbodiimide method with various modifications. In a two-step procedure the carboxy groups of the porphyrin were first activated by carbodiimide in aqueous solution or in organic media and then allowed to react with protein amino groups in slightly alkaline aqueous solution. A drawback of the methods where carbodiimides are used for activation of porphyrins having more than one carboxy group, is that a mixture of different products is formed as a result of the carbodiimide activation. The mixture comprises porphyrins having from 0 to n pieces of activated carboxy groups where n is the number of carboxy groups in the porphyrin before the activation. Multi-point activation of porphyrins often causes significant crosslinking and inactivation of biomolecules such as proteins when they react with such reagents.
An additional drawback of the carbodiimide chemistry is that it does not allow the active product to be isolated in pure form, but the product remains in form of undefined mixture. Furthermore, the active carbodiimide adduct is instable. It is sensitive to hydrolysis as well as to many other nucleophiles. Typically, the carbodiimide adducts react intramolecularly producing non-reactive N-acylurea. The chemical instability of the carbodiimide-activated porphyrin adducts makes their use difficult and unreliable. Also porphyrin-antibody conjugates prepared by carbodiimide mediated coupling has been found to exhibit poor storage s

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