Organic compounds -- part of the class 532-570 series – Organic compounds – Four or more ring nitrogens in the bicyclo ring system
Reexamination Certificate
2001-02-16
2002-08-27
Riley, Jezia (Department: 1656)
Organic compounds -- part of the class 532-570 series
Organic compounds
Four or more ring nitrogens in the bicyclo ring system
C435S006120
Reexamination Certificate
active
06441167
ABSTRACT:
This invention relates to novel compounds that can complex with lanthanide cations processes for their preparation and the use of the resulting lanthanide chelates as biomolecular probes. In particular, this invention relates to complexing compounds which contain novel photosensitizers and can produce long-lived fluorescence for use in bioaffinity assays, especially those of HTRF (homogeneous time-resolved fluorescence).
With the growth of combinatorial chemistry and high-throughput screening, particularly within the pharmaceutical industry, the requirement for biological assays has dramatically increased. Traditional assay technologies, which are often based on radioisotope labels, are unable to achieve the desired throughput whilst simultaneously reducing assay volumes. As a result of the deficiencies inherent in traditional methodologies there has been a shift towards the use of new technologies based on fluorescence. Such techniques can have a number of advantages over radioactive assays, but ability to automate, ease of use, miniaturisability, and sensitivity are of particular importance. One of the primary technologies utilised is homogeneous time resolved fluorescence energy transfer (HTRF). This proximity based method requires the use of a fluorescent donor moiety covalently attached to the interacting molecules, either directly or via labelled antibodies or labelled streptavidin which, when in proximity with a second fluorescent or chromophoric label (the acceptor), leads to a modulation of the fluorescence properties of the donor. Such methods provide useful information about the structure, conformation, relative location and/or interactions of macromolecules. In particular. HTRF has widespread application in high throughput screening of molecular interactions and enzymes using proteins, ligands and substrates labelled with donors and acceptors.
Traditional fluorescent labels of organic dyes such as fluoresceins and rhodamines have long been employed as bioanalytical tools in immunoassays. Lanthanide chelates are more recently developed fluorescence agents and have been found to possess properties which make them very suited as potential labels in the bioassay field. Thus, the lanthanide chelates are capable of giving long-lived and longer wavelength fluorescent emissions upon excitation. Through time-delay measurements they have demonstrated clear advantages over conventional fluorescent labels in terms of experiencing less quenching and background interference while exhibiting increased detection sensitivity. In addition to these advantages, many lanthanide chelates have improved solubility properties and are able to efficiently transfer energy from their excited states to neighbouring acceptor molecules. As such they are ideal agents for HTRF use especially for developing high-throughput automated and miniaturized binding assays with the inclusion of immunoassays, DNA hybridization assays, receptor binding assays, enzyme assays. cell-based assays. immunocytochemical or immunohistochemical assays.
Lanthanide chelates typically comprise a chelating group which binds the lanthanide and an organic sensitiser group. The sensitiser group has the function of absorbing light and transferring energy to the lanthanide. It thereby overcomes the inherently low absorbance of the lanthanide ions. Such chelates have been extensively reviewed, for example in Li and Selvin (J. Am. Chem. Soc (1995) 117, 8132-8138). Lanthanide chelator groups comprising a plurality of polyaminocarboxylate groups are commonly used. European patent EP0203047B1 discloses fluorescent lanthanide chelates comprising “TEKES” (4-(4-isothio-cyanatophenylenthynyl-2,6-{N,N-bis(carboxymethyl)aminomethyl]-pyridine) type photosensitizers. Patent application WO 96/00901A1 discloses lanthanide chelates comprising the chelator group DTPA (diethylenetriaminepentacetic acid) covalently bonded to a coumarin or quinolone-like sensitisers. Heyduck and Heyduck (Anal. Biochem. (1997)
248, 216-227) describe compounds of similar structure to those of WO
96/00901 but differ in that they possess a thiol-reactive pyridyl disulphide moiety which allows covalent attachment to macromolecules.
It is widely recognised that the role of the sensitiser group is of fundamental importance in that they impart to the chelates different 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. It is advantageous to have a diversity of novel fluorescent probes to use and develop HTRF assays. There is consequently a need for more and better ways of fluorescently labelling assay components.
The present invention therefore provides, in a first instance, a lanthanide chelate comprising one or more sensitiser group(s) covalently attached to a lanthanide chelating group which is characterised in that the sensitiser group is a group of formula (I)
where X is a group that couples the said sensitiser group to the said chelating group.
Suitably X is any group that is capable of covalently linking the sensitiser group with the chelator group and, at the same time, does not affect the ability of the chelating group to bind the lanthanide cation. Preferably X is a group —NH(CH
2
)pNH— in which p is 2, 3 or 4 and which forms an amide bond with the chelating group. Most preferably X is a group —NH(CH
2
)
2
NH—.
Preferably the lanthanide chelate contains 1 or 2 sensitiser group(s) of formula (I).
Where used herein the term [lanthanide] chelating group is used to describe a group that is capable of forming a high affinity complex with lanthanide cations such as Tb
3+
, Eu
3+
, Sm
3+
, Dy
3+
. Any fluorescent lanthanide metal can be used in the chelates of this invention but it is expected that chelates containing europium or terbium will possess the best fluorescent properties. Most preferably the lanthanide metal is europium.
Suitable examples of chelating groups include those described in WO 96/00901. Preferably the chelating group will be either DTPA (diethylenetriaminepentacetic acid) or TTHA (triethylenetetraaminehexacetic acid), that is to say compounds of the formula (II)
where n=1 (DTPA) or n=2 (TTHA). Both DTPA and TTHA are well known in the art and are available from commercial suppliers. Alternatively the chelating group is a compound of formula (III).
A compound of formula (III) may be prepared by reaction of the corresponding N,N-&agr;-bis(carboxymethyl)-L-lysine with isophthalic acid activated by O—(N-succinimidyl)-1,1,3,3-tetramethyluronium tetrafluoroborate.
Typically a compound of formula (IV)
in which L is a group —NH(CH
2
)pNH
2
where p is 2, 3 or 4 would be used in the preparation of a lanthanide chelate of formula (I). A compound of formula (IV) can be prepared from Cinoxacin (commercial product supplied by Sigma) by forming its acid chloride derivative and then reacting with an appropriate alkylene diamine reagent. A compound of formula (IV) in which p is 3 or 4 is believed to be novel.
Compounds of structure (I) have desirable spectral properties in solution, but to use them in a biological assay it is necessary to attach them to the molecules such as proteins, nucleic acids, lipids, carbohydrates or peptides. Reagents containing reactive groups suitable for derivatising macromolecules will also form part of the invention. The invention therefore provides, in a further aspect, for a lanthanide chelate of structure (I) further comprising a linker group wherein the linker group is either a group of formula (V)
in which Y is CH
2
, CH
2
CH
2
or —CH
2
CH(COOH)— and R
2
is a reactive group which is suitable for derivatising macromolecules; or the linker group is a group of formula (VI)
R
2
—(CH
2
)
n
—Z—NH— (VI)
in which n is 1 to 5, Z is a bond or a group —CH
2
CH(COOH)— and R
2
is as defined for formula (V).
For groups of formula (V) and (VI) the point of attachment to the chelatin
Leach Colin Andrew
Moore Keith James Millan
Stanway Steven James
Kinzig Charles M.
Riley Jezia
SmithKline Beecham plc
Stercho Yuriy P.
Venetianer Stephen
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