Organic compounds -- part of the class 532-570 series – Organic compounds – Heterocyclic carbon compounds containing a hetero ring...
Reexamination Certificate
2001-06-20
2004-05-25
Zucker, Paul A. (Department: 1621)
Organic compounds -- part of the class 532-570 series
Organic compounds
Heterocyclic carbon compounds containing a hetero ring...
C546S194000, C549S393000, C552S236000, C552S238000, C562S565000, C436S172000, C206S569000
Reexamination Certificate
active
06740756
ABSTRACT:
FIELD OF INVENTION
The present invention relates to the identification and preparation of organic agents that can complex lanthanide cations. In particular, this invention relates to complexing agents which contain novel photosensitizers and can produce long-lived fluorescence for use in bioaffinity assays, especially HTRF (homogeneous time-resolved fluorescence) assays.
BACKGROUND OF THE INVENTION
A wide variety of bioassays are used in the pharmaceutical industry to identify drug development candidate compounds. Recent advances in the identification of pharmaceutical targets, together with the vastly increased output of new compounds using techniques such as combinatorial chemistry have created a need to increase bioassay throughput (number of samples measured per unit time) drastically to meet discovery objectives. Robotics, miniaturization and homogeneous assay formats have all been incorporated into high throughput screening (HTS) assays to increase throughput. Ideally, an analytical technique suitable for both miniaturization and homogeneous assay formats must provide maximal detection sensitivity and interaction in situ, while requiring only minimal assay time and liquid handling (e.g., separation and filtration). Present analytical techniques, such as those which use radiolabels, are unsatisfactory for HTS use because they lack sensitivity, require large sample size and manual liquid handling.
Compared to traditional radiolabels, fluorescent labels have more desirable lifetime, solubility and sensitivity properties for use in HTS assays. The unique lifetime properties of fluorescent labels also meet the needs of fluorescence polarization (FP) and fluorescence correlation spectroscopy (FCS) in the investigation of slow rotational and translational changes in macromolecules.
Traditional fluorescent labels such as organic dyes, e.g., fluoresceins and rhodamines, have long been employed as bioanalytical tools in immunoassays. More recently, lanthanide chelates have been developed as fluorescence agents for use in the bioassay field. These lanthanide chelates have been reviewed. See Dickson, J.
Photochemistry and Photobiology
, 27(1995) 3-9; and Mathis,
J. Clinical Ligand Assay
20 (1997) 141-145.
The lanthanide chelates are capable of producing long-lived and long wavelength fluorescent emissions upon excitation. In time-delay measurements, they have demonstrated clear advantages over conventional fluorescent labels, in particular less quenching and background interference, while exhibiting increased detection sensitivity. In addition to these advantages, many lanthanide chelates have demonstrated superior solubility properties and are able to efficiently transfer energy from their excited states to neighboring acceptor molecules. These advantages render lanthanide chelates ideal agents for HTRF use, especially for developing high-throughput automated and miniaturized binding assays, inncluding immunoassays, DNA hybridization assays, receptor binding assays, enzyme assays, cell-based assays, immunocytochemcial or immunohistochemical assays.
A number of lanthanide (e.g. terbium, europium) complexes are known, but only three classes of lanthanide chelates, exemplified by the compounds shown in Table I below, are considered to be useful in HTRF:
TABLE I
Cryptates (Packard) bipyridine type; i.e.
DTPA Chelates (Berkeley) diethylenetriamine-pentaacetic acid type; i.e.
PMDA Chelates (Wallac) pyridylmethylamine-diacetic acid type; i.e.
These chelates haven been described as having chemical stability, long-lived fluorescence (greater than 0.1 ms lifetime) after bioconjugation and significant energy-transfer in specific bioaffinity assay U.S. Pat. No. 5,162,508, issued to Lehn, et al. on Nov. 10, 1992 discloses bipyridine cryptates. Polycarboxylate chelators with TEKES type photosensitizers (EP 0203047 A1) and terpyridine type photosensitizers (EP 0649020 A1) are known. International Publication No. WO 96/00901 of Selvin et al., having an International Publication Date of Jan. 11, 1996, discloses diethylenetriaminepentaacetic acid (DTPA) chelates winch used carbostyril as sensitizer. Baley, et al.,
Analyst,
109, (1984) 1449; Ando, et al.
Biochim. Biophys. Acta
, 1102, (1992) 186; and Heyduk et al.,
Anal. Biochemistry
, 248, (1997) 216 also describe DTPA lanthanide chelates which contain different sensitizers. Additional DTPA chelates with other sensitizers and other tracer metal are known for diagnostic or imaging use (e.g., EP 0450742 A1).
The lanthanide chelates provided by the present invention include novel sensitizers which differ from carbostyril and other known chelates. More specifically, these novel sensitizers impart onto the present chelates advantageous physicochemical properties pertaining to excitation wavelength, lifetime, quantum yield, quenching effect, complex stability, photostability, solubility, charge, nonspecific protein interaction, biocoupling efficiency and ease of preparation. Such advantages are desirable to provide a diversity of novel fluorescent probes for use in, and development of, HTRF assays.
SUMMARY OF THE INVENTION
An object of the present invention is to provide novel lanthanide chelate compounds, and a method for using such compounds in fluorescence detection-based techniques or bioassays.
Accordingly, in the first aspect, this invention provides a compound according to Formula I.
In still another aspect, this invention provides a method for using the compounds of Formula I in fluorescence detection-based techniques or bioassays.
In yet another aspect, this invention provides a kit for fluorescence detection-based techniques or bioassays which use the compounds of Formula I as the basis for signal detection and measurement.
DETAILED DESCRIPTION OF THE INVENTION
Each compound of the present invention comprises four functional parts: a lanthanide metal cation (e.g. Tb III, Eu III, Sm III, Dy III), a chelator for the lanthanide metal, a photosensitizer for photoexcitation and energy transfer, and a linker for bioconjugation to the target biomolecule, that is, the biomolecule being measured using a fluorescence detection-based spectroscopic technique or bioassay.
The present invention provides compounds of Formula I:
wherein:
[\N&Lgr;]
n
is a chelator selected from the group consisting of: diethylenetriaminepentaacetic acid (DTPA) (n=1) or triethylenetetraaminehexaacetic acid (TTHA) (n=2) or a polyaminocarboxylate derivative of DTPA or TTHA, preferably DTPA, which chelates a lanthanide metal cation, preferably selected from the group consisting of: Tb III, Eu III, Sm III, and Dy III.
The sensitizer R1 is usually related to an aromatic or heteroaromatic amine whose chromophore plays a vital role in excitation and energy transfer. Superior sensitizers usually have highly conjugated systems and an added capacity for lanthanide complexation. We have found several sensitizers, belonging to two structural classes—phenones and quinolines—that provide highly fluorescent compounds of Formula I. R1 is more preferably selected from the following group: aminoacetophenones (AAP), aminobenzophenones (ABP), aminofluorenones (AF), aminoxantones (AX), amino-azaxanthones (AAX), aminoanthraquinones (AAQ), and aminoacridones (AAC):
wherein for each nucleus, the amino group NH
2
may be attached at one of any possible positions on the phenyl ring. The point of amide attachment to the chelator [\N&Lgr;]
n
in Formula I may similarly be attached at one of any possible positions on the phenyl ring. R3 and R4 are independently selected from the group consisting of: H, OH, NH
2
, COCH
3
, COPh, OPh, NHPh, CN, NO
2
, CO
2
H, CO
2
CH
3
, I, Br and Cl.
Sensitizers of the present invention belonging to the quinoline class can be further categorized into 3-aminoquinolines (3AQ), and 6-aminoquinolines (6AQ). Preferably in the quinoline compounds of the present invention, R1 is selected from the group consisting of:
wherein R3 and R4 are as defined herein above.
The linker R2 is an amine or other moiety having a functional
Chan George Wai-Kin
Hertzberg Robert P.
Henderson Loretta J.
Kinzig Charles M.
SmithKline Beecham Corporation
Zucker Paul A.
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