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

Abstract

The invention relates to new fluorescent dyes of formula (I) 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 AIDA 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. 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 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-spectral range 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 AIDA-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 one- and twodimensional single molecule spectroscopic techniques (SMS). 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.

1 . Compounds 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 , —CONH(CH 2 ) n OH, wherein n=2 - 8, —CONR 8 R 9 , —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, which could be 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 which could be 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 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,  
 R 7  is a commonly used carboxyl protecting or carboxyl activating group  
 R 8  or R 9  is hydrogen and the other is lower alkyl (C 1 -C 4 ), phenyl, benzyl, or R 8  and R 9  are part of a 5 or 6 membered ring,  
 R 10  and R 11  are independently hydrogen or (C 1 -C 4 )alkyl  
 R 12  is (C 1 -C 10 )alkyl, phenyl, which both can be substituted by (C 1 -C 4 ) alkyl, protected amino group or halogen.  
 
     
     
         2 . Compounds according to    claim 1    represented by the following structures:  
                 
 
     
     
         3 . Compounds represented by formula (II-III)  
       A-B-D-C-D′-E   (Formula (II)) A-B-D-E-D′-C   (Formula (III))  
       wherein 
 A is a solid support selected from standard materials applied in solid phase and solution phase organic chemistry,  
 B is a linker allowing cleavage of fluorescent conjugates of formula (II-III) for liberation of the D-C-D′-E or D-E-D′-C fragment, respectively,  
 C is a compound selected from formula (I)  
 D and D′ are independently a bond or a spacer selected from α,ω-diamino-alkanes, diaminocyclohexyl, bis-(aminomethyl)-substituted phenyl, α-amino-ω-hydroxyalkanes, alkylamines, cyclic alkylamines or cyclic alkyldiamines or amino acids without or with additional functionality in the side chain,  
 E is the molecule to be investigated.  
 
     
     
         4 . Compounds according to    claim 3    wherein 
 A is selected from functionalized polystyrene based resins, polyacrylamide based polymers, polystyrene/polydimethylacrylamide composites, PEGA resins, polystyrene-polyoxyethylene based supports, Tentagel, PEG-polystyrene graft polymeric supports, glass surfaces, functionalized surfaces, materials grafted with functionalized surfaces, or polyethylenglycol,  
 B is selected from benzyl, benzhydryl, benzhydryliden, trityl, xanthenyl, benzoin, silicon, or allyl based linkers,  
 C is a compound selected from formula (I)  
 E is a low molecular weight compound, a peptide, a protein, a carbohydrate, a nucleic acid, or a lipid containing a functional group for conjugate formation.  
 
     
     
         5 . Compounds according to    claim 3    represented by the following structures:  
                 
 
                 
 
     
     
         6 . Compounds represented by formula (IV):  
       E-D′-C   (Formula (IV))  
       wherein 
 E is the molecule to be investigated  
 D′ is a bond or a spacer selected from α,ω-diamino-alkanes, diaminocyclohexyl, bis-(aminomethyl)-substituted phenyl, α-amino-ω-hydroxy-alkanes, alkylamines, cyclic alkylamines, cyclic alkyldiamines or amino acids without or with additional functionality in the side chain  
 C is a compound selected from formula (I).  
 
     
     
         7 . Compounds according to    claim 6    represented by the following structures:  
                 
 
     
     
         8 . A method for identification of an interaction between an AIDA labelled molecule and a binding molecule in homogeneous solution wherein the method comprises the following steps: 
 Step 1A: Providing an AIDA labelled molecule selected from formula (IV)    Step 1B: Admixing the AIDA-labelled molecule of formula (IV) with a binding molecule; and then    Step 1C: selectively detecting a binding event with the AIDA-labelled molecule described in Step 1B and the binding molecule by methods of fluorescence spectroscopy.    
     
     
         9 . Method according to    claim 8    wherein the methods of fluorescence spectroscopy are measurements of 
 Increase of fluorescence anisotropy/polarisation of AIDA emission in continuos wave=prompt=steady state fluorometers,  
 Increase of rotational correlation time in time-resolved fluorescence equipments  
 Increase in translation diffusion time in single molecule fluorescence experiments determined from autocorrelation calculations on the time trace of fluorescence fluctuations,  
 Increase or decrease of AIDA fluorescence emission in the wavelength range between 350 and 700 nm with excitation wavelengths in the range between 300 and 400 nm,  
 Fluorescence resonance energy transfer (donor quenching or acceptor sensitisation) from excited tryptophan (donor) in the binding molecule which in this case is a peptide or protein to the AIDA dye (acceptor) in the molecule of the conjugate,  
 Fluorescence resonance energy transfer (donor quenching or acceptor sensitisation) from the excited AIDA dye in the conjugate molecule (donor) to a fluorescent label (acceptor) of the binding molecule which in this case can comprise any compound class.  
 
     
     
         10 . A method for identification of an interaction between an AIDA labelled molecule on the solid support which is conventionally used in solid phase organic chemistry and a binding molecule in homogeneous solution containing the solid support wherein the method comprises the following steps: 
 Step 2A: Providing an AIDA labelled molecule as conjugate of formula (II or III)    Step 2B: Admixing the AIDA-labelled molecule as conjugate of formula (II or III) with a binding molecule; and then    Step 2C: selectively detecting a binding event with the AIDA-labelled molecule described in Step 2B and the binding molecule by methods used in fluorescence spectroscopy resulting in a quantitative signal providing a means to identify the AIDA-linked molecule with the highest binding affinity to the binding molecule,    Step 2D: Isolation of the solid support containing the identified AIDA-molecule represented by formula (II or III)    Step 2E: Selectively detecting a binding event with the AIDA-labelled molecule described in Step 2D and the binding molecule by various methods used in fluorescence spectroscopy described in the procedure 1A-C.    
     
     
         11 . Method according to    claim 10    wherein the fluorescence spectroscopic methods in step 2C are 
 Direct detection of binding of fluorescently labelled macromolecules to AIDA containing solid supports applying confocal microscopic and spectroscopic techniques  
 measurement of enhancement of the change in molecular brightness by chemically linking AIDA to a second environmentally sensitive molecule as commonly used in conventional fluorescence spectroscopy performed during the synthesis of the compound on the solid support,  
 measurement of fluorescence resonance energy transfer: From AIDA to a suitable long wavelength dye which will thereby be sensitised using AIDA UV-excitation detected by change in molecular brightness at the emission wavelength of the long wavelength dye,  
 measurement of fluorescence resonance energy transfer: Reduction of specific brightness of AIDA on the molecule linked to the solid support at 351 nm excitation and 400 nm emission wavelengths,  
 Detection of the change in quantum yield by measuring reduction or increase in molecular brightness by time-resolved single molecule spectroscopy.