Fluorescent dyes (AIDA) for solid phase and solution phase...

Details Fluorescent dyes (AIDA) for solid phase and solution phase... Fluorescent dyes (AIDA) for solid phase and solution phase...

1998-12-21

2001-03-27

Lambkin, Deborah C. (Department: 1626)

Organic compounds -- part of the class 532-570 series

Organic compounds

Nitrogen attached directly or indirectly to the purine ring...

C544S229000, C548S110000, C548S361100

Reexamination Certificate

active

06207831

ABSTRACT:

TECHNICAL FIELD
The present invention relates to the field of ultra high-throughput screening on the solid support and in homogeneous solution by a novel generic labelling technology. The new labelling technology is based on new chemically stable fluorophores, which possess reactive chemical functionalities for attachment to a solid support and subsequent start of combinatorial synthesis of compound libraries.
BACKGROUND
Three new scientific disciplines show the highest promise of fulfilling the need for increased predictability and for lowering the overall attrition rate of the drug discovery process. (1) Functional Genomics was invented to generate new innovative molecular targets. (2) Combinatorial Chemistry provides increasingly efficient ways to generate molecular diversity with which to probe the targets. (3) High-throughput screening (HTS) plafforms provide efficiency and quality in finding potential lead compounds. High throughput screening within most pharmaceutical companies currently involves performing several million assays per year. Meanwhile, HTS has become a discrete discipline assimilating biochemistry, biophysics and cell/molecular biology combined with detection/liquid handling technologies and automation processes. With all the phantastic oportunities these new scientific disciplines offer, it became already clear, that the time consuming process in functional genomics is the identification of the physiological function of a new protein. Whereas combinatorial chemistry approaches help to synthesize a multifold of compounds during the time needed for classical synthesis, it is known now that between 5 and 10 times the number of assays are needed to identify hit compounds from screens. The challenge the community of applied science is currently faced with, is to fully exploit the advantages of (1) and (2) in a timely manor by speeding up the process of synthesis, protein function identification and screening. The current invention opens a new possibility for integrating the advantages of combinatorial chemistry and genomics with HTS by providing the efficiency needed for screening compounds directly on the solid support. Target macromolecules of even unknown functionality can be tested for their direct binding affinity to compounds of interest. The new fluorescent chemistry, generically described as AlDA-chemistry in the following, is suitable also to screening assays in homogeneous solution, either by direct application of the compounds conjugated to the AlDA chemistry or by cleavage of the AlDA conjugates from the solid support by well known chemical or photophysical means. After the release of the AlDA conjugated “binder” identified in a solid-phase screening technology, the affinity to the macromolecule of interest can be determined by conventional ensemble averaging fluorescence spectroscopic techniques in assay volumes used in microtiter plates. In addition, single molecule spectroscopic techniques performed in microliter volumes and applied in so called nanocarriers can be used.
SUMMARY OF THE INVENTION
The invention refers to specific fluorescent dyes, which can be used in high throughput screening both, on the solid phase as well as in homogeneous solution. The new fluorescent dyes generically referred to as AlDA chemistry is suitable for various methods of solid phase and solution phase organic chemistry for synthesis of molecules to be investigated for therapeutic use in disease states. As used herein, “AlDA” is an abbreviation for arylindazol compounds or derivatives described herein. The molecules of therapeutic interest can be synthesized as fluorescent conjugates by two methods: (a) a solid support is loaded with a cleavable linker (acid-, base-, redox- or light sensitive) to which initially the fluorescent dye is attached. The dyes possess a second functionality, which serves as attachment point for spacer elements. The spacer bears a further functional group which is used as starting point of the synthesis of the molecules to be investigated; (b) the fluorescent dye can also be introduced as end-cap in the last synthesis step of a reaction sequence. The specific dyes described in the invention are chemically stable under a broad range of reaction conditions usually applied in solid phase and solution phase organic chemistry. The conjugates emit fluoresence in the visible and UV-spectrum on excitation at wavelengths of their absorption. These fluorescence properties allow for multiple applications in fluorescence based processes for the identification of inhibitors of molecular interactions and for the identification of molecules which bind to target macromolecules like peptides proteins, nucleic acids, carbohydrates etc. The fluorescence detection technologies used for monitoring binding of AlDA-conjugated compounds to macromolecules include conventional macroscopic techniques (ensemble averaging) which detect changes in fluorescence intensity, anisotropy(polarization), fluorescence resonance energy transfer, fluorescence lifetime, rotational correlation time as well as single molecule spectroscopic techniques (SMS). SMS include excitation by one or two laser wavelengths including laser lines at 325 nm, 351 nm, 453 nm, 488 nm, 514 nm, 543 nm, 632 nm and other excitation possibilities . The fluorescence light emitted from single molecules diffusing through a confocal focus as applied in single molecule spectroscopy passes through one or two filters, and polarisers before the light reaches the avalanche photodiode detector. The comprehensive detection technologies in SMS to be applied to AlDA include translational diffusion, rotational diffusion, fluorescence lifetime, fluorescence brightness, spectral shifts, fluorescence energy transfer, triplet transition probabilities and multiplex detection. The dyes show sufficient stability in chemical transformations, have little triplet state formation and are not photoreactive under the conditions used for detection of binding events to biomolecules, making them to an excellent tool for combination of combinatorial chemistry (solid phase and solution phase chemistry) and biological investigations (ultra high throughput screening).
Uses of the dye include solid phase and solution phase organic chemistry, low molecular weight compound labelling, peptide labelling, protein labelling, optical spectroscopy and fluorescence. Synthesis of functionalized dyes and of dye conjugates (on solid support and in solution) are disclosed.
DETAILED CHEMICAL ASPECTS OF THE INVENTIONS
A first aspect of the invention is directed to a fluorescent dye represented by formula (I)
Wherein one of the radicals R
1
or R
2
and one of the radicals R
3
or R
4
is hydrogen and the other is independently —COOH, —COOR
7
, —CONH
2
, —CONR
8
R
9
, —CONH(CH
2
)
n
OH, wherein n=2-8, —CH
2
OH, —CH
2
NH
2
, —NO
2
, NR
10
R
11
, NHCOR
12
, Cl, Br, F, —CF
3
, O(C
1
-C
4
)-alkyl (optionally substituted by methyl or phenyl at any of the carbons C
1
-C
4
), —N=C=O, N=C=S, —SO
3
H, —SO
2
NH(CH
2
)
n
NH
2
, (C
1
-C
4
) alkyl, (C
1
-C
16
)-alkyl substituted at the terminal carbon with —COOH, —COOR
7
, —CONH
2
, —CONR
8
R
9
, —CONH(CH
2
)
n
OH, wherein n=2-8, —CH
2
OH, —CH
2
NH
2
, —N=C=O, N=C=S, —SO
3
H, —SO
2
NH(CH
2
)
n
NH
2
, —CONH(CH
2
)
n
NH
2
, wherein n=2-8, and the NH
2
-group could also be substituted by (C
1
-C
4
) alkyl or a commonly used amino protecting group such as tert-butyloxycarbonyl, 9-fluorenylmethoxycarbonyl, phthalimido, trifluoroacetamido, methoxycarbonyl, ethoxycarbonyl, benzyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl,
and one of the radicals R
5
or R
6
is hydrogen and the other is hydrogen, halogen, O(C
1
-C
4
)-alkyl (optionally substituted by methyl or phenyl at any of the carbons C
1
-C
4
), —NO
2
, NR
10
R
11
, NHCOR
12
, (C
1
-C
4
) alkyl, (C
1
-C
16
)-alkyl substituted at the terminal carbon with —COOH, —COOR
7
, —CONH
2
, —CONR
8
R
9
, —CONH(CH
2
)
n
OH, wherein n=2-8, —CH
2
O

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