Labels, their production process and their uses

Abstract

Labels are disclosed capable of forming a covalent or non-covalent bond with a target molecule, particularly a biological molecule. The structure of these labels may consist of a dye covalently bound by one or more carbons on its chemical structure to one or more [FUNC] group(s), and optionally one or more [SOL] group(s). The structure of these labels allow selection of dyes from a wide variety of different excitation and emission wavelengths and allow easy functionalization of the dye without appreciably altering its spectral characteristics or its solubility characteristics.

1 .- 23 . (canceled) 
     
     
         24 . A label consisting of a dye comprising, a carbon covalently bonded to:
 one or more [FUNC] group(s), and   optionally one or more [SOL] group(s),   said label having the following formulae (9) or (10):   
       
         
           
           
               
               
           
         
       
       each of Q′ 1 , Q′ 2 , Q′ 4 , Q′ 5 , Q′ 7 , Q′ 8 , Q′ 10  and Q′ 11  each independently from each other represents hydrogen, hydroxyl, halogen, acetyl, amine, substituted amine, quaternary ammonium, phosphate, nitro, carboxylic acid and its salts, sulphonic acid and its salts, alkylcarboxy with 2 to 30 carbon atoms, linear or branched alkyl having 1 to 30 carbon atoms, cycloalkyl having 3 to 14 carbon atoms, alkyloxy having 1 to 30 carbon atoms, halogenoalkyl having 1 to 30 carbon atoms, hydroxyalkyl having 1 to 30 carbon atoms, alkylester having 2 to 40 carbon atoms, nitroalkyl having 1 to 30 carbon atoms, carboxyalkyl having 2 to 30 carbon atoms, aminoalkyl having 1 to 30 carbon atoms, sulphoalkyl having 1 to 30 carbon atoms, aryl, aryloxy, arylalkyl, halogenoaryl, arylester, [SOL] or [FUNC],
 Q′ 3 , Q′ 6 , Q′ 9  and Q′ 12  not being able to represent aryl or aryloxy, providing that at least one of Q′ 1  to Q′ 12  represents H or an aromatic ring, and providing that at least one of Q′ 1 , Q′ 2 , Q′ 4 , Q′ 5 , Q′ 7 , Q′ 8 , Q′ 10  and Q′ 11  represents [FUNC]; 
 
       
         
           
           
               
               
           
         
       
       in which each of D′ 1  to D′ 16 , each independently from each other represents hydrogen, hydroxyl, halogen, acetyl, amine, substituted amine, quaternary ammonium, phosphate, nitro, carboxylic acid and its salts, sulphonic acid and its salts, alkylcarboxy with 2 to 30 carbon atoms, linear or branched alkyl having 1 to 30 carbon atoms, cycloalkyl having 3 to 14 carbon atoms, alkyloxy having 1 to 30 carbon atoms, halogenoalkyl having 1 to 30 carbon atoms, hydroxyalkyl having 1 to 30 carbon atoms, alkylester having 2 to 40 carbon atoms, nitroalkyl having 1 to 30 carbon atoms, carboxyalkyl having 2 to 30 carbon atoms, aminoalkyl having 1 to 30 carbon atoms, sulphoalkyl having 1 to 30 carbon atoms, aryl, aryloxy, arylalkyl, halogenoaryl, arylester, [SOL] or [FUNC],
 providing that at least one of D′ 1  to D′ 16  represents H, and providing that at least one of D′ 1  to D′ 16  represents [FUNC]; 
 [FUNC] each representing independently an —X-A-Z group, in which:
 X is chosen from the group consisting of an oxygen atom, a sulphur atom, an NR 1 R 2 group, R 1  and R 2  each being independently of each other a hydrogen atom or a linear or branched C 1 -C 30 , preferably C 1 -C 18  and more preferentially C 1 -C 5  alkyl group; 
 A is an alkylene group, or an alkylene-arylene group; 
 Z is a reactive chemical function chosen from the group comprising the functions carboxylic acid and its salts, sulfonic acid and its salts, acid anhydride, acid chloride, esters such as alkyl ester, p-nitrophenyl ester, succinimidyl ester, sulphosuccinimidyl ester, azido such as acyl azide, azidonitrophenyl, hydrazide, 3-acyl-1,3-thiazolidine-2-thione, amine, substituted amine, quaternary ammonium, isocyanate, isothiocyanate, hydrazine, phthalimido, maleimide, haloacetamide, monochlorotriazine, dichlorotriazine, mono- or dihalogenated pyridine, mono- or dihalogenated diazine, aziridine, thiol, sulphonyl chloride, vinylsulphone, disulphide, methanethiosulphonate, hydroxyle, phosphoramidite, epoxy, aldehyde, carbonate, glyoxal, imidazolyl; 
 
 [SOL] each representing independently an —X′-A′-Z′ group, in which:
 X′ is chosen from the group consisting of an oxygen atom, a sulphur atom, an NR 1 R 2  group, R 1  and R 2  each being independently of each other a hydrogen atom or a linear or branched C 1 -C 30 , preferably C 1 -C 18  and more preferentially C 1 -C 5  alkyl group; 
 A′ is an alkylene group or alkylene-arylene group; 
 Z′ is either a polar group chosen from the group comprising the groups carboxylic acid and its salts, sulphonic acid and its salts, substituted amine, quaternary ammonium, carbohydrate, glycol, hydroxyl, nitro, phosphate, or an apolar group chosen from the group comprising the linear or branched alkyl groups having 1 to 30 carbon atoms, cycloalkyl having 3 to 14 carbon atoms, alkyloxy having 1 to 30 carbon atoms, halogenoalkyl having 1 to 30 carbon atoms, hydroxyalkyl having 1 to 30 carbon atoms, alkylester having 2 to 40 carbon atoms, aryl, aryloxy, substituted arylalkyl, arylalkyl, halogenoaryl. 
 
 
     
     
         25 . The label according to  claim 24 , wherein the alkylene group is a cyclic, linear or branched hydrocarbon chain having two free bonds, comprising 1 to 30 carbon atoms and the arylene group is an aromatic group, having two free bonds, comprising one or more aromatic rings, optionally substituted. 
     
     
         26 . The label according to  claim 24 , wherein [FUNC] is chosen from
 —X—(CH 2 ) r —COOSu,   —X—(CH 2 ) r —COOSuSO 3 Na,   —X—(CH 2 ) r —COOH,   —X—(CH 2 ) r SO 3 H,   —X—(CH 2 ) r —SO 3 Na,   —X—(CH 2 ) r —COO—C 6 H 4 —NO 2 ,   —X—(C 6 H 4 )—(CH 2 ) r —COOSu,   —X—(CH 2 ) r —NHCOCH 2 I, —X—(CH 2 ) r —NCS,   —X—(CH 2 ) r —C 6 H 4 CH(CH 3 )—COOSu,   —N(CH 3 ) 2 —(CH 2 ) r —SO 3 H, and   —X—(CH 2 ) r —OP[N(iPr) 2 ][CH 2 CH 2 CN],   X being as defined in  claim 24 , r is an integer from 1 to 18, and Su representing the succinimidyl group.   
     
     
         27 . The label according to  claim 24 , wherein [SOL] is chosen from
 —X—(CH 2 ) r —SO 3 Na,   —X—(CH 2 ) r —SO 3 H,   —X—(CH 2 ) r —C 6 H 3 (NO 2 ) 2 ,   —X—(CH 2 ) r —CH 3 , and   X being as defined in  claim 24  and, r is an integer from 1 to 18.   
     
     
         28 . The label according to  claim 24 , wherein said label is chosen from the group consisting of the structures (6), (7), (11) or (12): 
       
         
           
           
               
               
           
         
       
       in which each of y and z, identical or different, is an integer equal to 1, 2, 3, 4, 5, 6, 7 or 8
 Z represents —COOH or 
 
       
         
           
           
               
               
           
         
         Y represents SO 3   −  or SO 3 Na 
       
       
         
           
           
               
               
           
         
       
       in which each of y and z, identical or different, is an integer equal to 1, 2, 3, 4, 5, 6, 7 or 8
 Z represents —COOH or 
 
       
         
           
           
               
               
           
         
         Y represents SO 3   −  or SO 3 Na. 
       
     
     
         29 . A process for preparing labels as defined in  claim 24 ,wherein said process comprises a nucleophilic substitution reaction between either:
   Z-A-L and [DYE′]-Nu
     or:     [DYE″]-L and Z-A-Nu
   Z and A being as defined in  claim 24 ;   L representing a leaving group;   Nu representing a nucleophilic group chosen from the group comprising —OH, —SH or —NR 1 R 2 , R 1  and R 2  each being independently of each other a hydrogen atom or a linear or branched C 1 -C 30  alkyl group;   [DYE'] and [DYE″] representing the dye formula (4) or (5):   
       
         
           
           
               
               
           
         
       
       in which:
 Q 1  to Q 12  and D 1  to D 16 , each independently represents hydrogen, hydroxyl, halogen, acetyl, amine, substituted amine, quaternary ammonium, phosphate, nitro, carboxylic acid and its salts, sulphonic acid and its salts, alkylcarboxy with 2 to 30 carbon atoms, linear or branched alkyl having 1 to 30 carbon atoms, cycloalkyl having 3 to 14 carbon atoms, alkyloxy having 1 to 30 carbon atoms, halogenoalkyl having 1 to 30 carbon atoms, hydroxyalkyl having 1 to 30 carbon atoms, alkylester having 2 to 40 carbon atoms, nitroalkyl having 1 to 30 carbon atoms, carboxyalkyl having 2 to 30 carbon atoms, aminoalkyl having 1 to 30 carbon atoms, sulphoalkyl having 1 to 30 carbon atoms, aryl, aryloxy, arylalkyl, halogenoaryl, arylester,
 Q 3 , Q 6 , Q 9  and Q 12  not being able to represent aryl or aryloxy, providing that at least one of Q 1  to Q 12  represents H or an aromatic ring, and, 
 providing that at least one of D 1  to D 16  represents H. 
 
 
     
     
         30 . The process according to  claim 29 , wherein the nucleophilic substitution reaction is a Williamson reaction in the presence of a base. 
     
     
         31 . The process according to  claim 29 , wherein the leaving group L is chosen from the group consisting of a halogen, a methanesulphonate, para-toluenesulphonate group and a diazonium group. 
     
     
         32 . The process according to  claim 29 , wherein [DYE′]-Nu is obtained by the following successive reactions:
 a) nitration of [DYE′]; 
 b) reduction of the product obtained in step a); 
 c) optionally: either alkylation of the product obtained in step b), or diazotization of the product obtained in step b) in order to obtain a diazonium salt then nucleophilic substitution on this diazonium function. 
 
     
     
         33 . The process according to  claim 29 , wherein [DYE]-L is obtained by the following successive reactions:
 a) nitration of [DYE″];   b) reduction of the product obtained in step a);   c) diazotization of the product obtained in step b) in order to obtain a diazonium salt;   d) optionally: nucleophilic substitution on the diazonium function of the product obtained in step c).   
     
     
         34 . The process according to  claim 29 , wherein said process comprises a nucleophilic substitution reaction between either:
   Z′-A′-L and [DYE′]-Nu
     or:     [DYE″]-L and Z′-A′-Nu
   Z′ and A′ being as defined in  claim 24 ;   L, Nu, [DYE′] and [DYE″] being as defined previously in  claim 29 ;   [DYE′]-Nu and [DYE″]-L being prepared according to the process of  claim 29 .   
     
     
         35 . A process of labelling a target molecule comprising the step of coupling said target molecule with a label according to  claim 24 . 
     
     
         36 . A method for labelling biological molecules comprising the step of coupling said biological molecule with a label according to  claim 24 . 
     
     
         37 . The method for labelling biological molecules according to  claim 36 , wherein the step of coupling is performed in the presence of a peptide coupling agent, for example carbodiimide-type reagents, IDDQ (1-isobutyloxycarbonyl-2-isobutyloxy-1,2-dihydroquinoline), phosphonium-type reagents, uronium-type reagents, Woodward reagents or Curtius reagents. 
     
     
         38 . The method for labelling biological molecules according to  claim 37 , wherein the coupling is carried out in the presence of dimethylformamide. 
     
     
         39 . A method for detecting a biological or non-biological molecule comprising the step of coupling a label according to  claim 24  with said biological or non-biological molecule, and the step of detecting said molecule coupled with the label by absorption spectrometry, fluorescence spectrometry, infrared spectrometry, electrophoresis, near infra-red or infra-red medical imaging. 
     
     
         40 . The label according to  claim 24 , wherein said label is selected from the group consisting of: