Oxazine derivatives

Abstract

Oxazine derivatives with which a broad spectrum of material to be studied can be labelled and identified by means of fluorescence techniques are described.

1 - 36 . (canceled)  
   
   
       37 . An oxazine derivative having the general formula (I)  
     
       
         
         
             
             
         
       
       wherein:  
       (a) R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9  and R 10  are radicals independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, alkenyl, alkenoxy, aryl, aryldiazo, alkylaryl, arylalkoxyl, alkoxy, alkoxycarbonyl, hydroxy, halogen, cyano, carbonyl, acyl, acyloxy, carboxyl, carbonamide, halogen carbonamide, sulfonyl, sulfonyl halide, acid ester, acid anhydride, acid halide, imide, imidyl ester, isothiocyanate, phosphoramidite, azide, dithionicotine derivative, amine and substituted derivatives thereof containing at least one radical substituent;  
       (b) R 1  with R 2  and/or R 9  with R 10  can each form a C3 or C4 bridge, wherein the bridge is unsaturated or saturated, and unsubstituted or substituted with at least one bridge substituent;  
       (c) the radical substituent and the bridge substituent are independently selected from the group consisting of alkyl, cycloalkyl, alkenyl, alkenoxy, aryl, aryldiazo, alkylaryl, arylalkoxyl, alkoxy, alkoxycarbonyl, hydroxy, halogen, cyano, carbonyl, acyl, acyloxy, carboxyl, carbonamide, halogen carbonamide, sulfonyl, sulfonyl halide, acid ester, acid anhydride, acid halide, imide, imidyl ester, isothiocyanate, phosphoramidite, azide, dithionicotine derivative and amine; and  
       d) at least one of the radicals R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9  and R 10  comprises or possesses at least one reactive group, or a salt thereof, adapted to bind to a material to be investigated.  
     
   
   
       38 . The oxazine derivative according to  claim 37 , wherein R 1  with R 2  and/or R 9  with R 10  form a saturated or unsaturated C4 bridge.  
   
   
       39 . The oxazine derivative according to  claim 38 , wherein R 1  with R 2  or R 9  with R 10  form a saturated or unsaturated C4 bridge.  
   
   
       40 . The oxazine derivative according to  claim 37 , wherein R 3 , R 4 , R 7  or R 8  comprises or possesses the at least one reactive group.  
   
   
       41 . The oxazine derivative according to  claim 40 , wherein the at least one reactive group is an acid ester, acid anhydride, acid halide, imide, imidyl ester, carboxyl, carbonamide, halogen carbonamide, sulfonyl halide, isothiocyanate, phosphoramidite, amine, azide, aryldiazo, aldehyde, ketone or dithionicotine derivative.  
   
   
       42 . The oxazine derivative according to  claim 41 , wherein the at least one reactive group is an acid ester, acid anhydride, acid halide, imide, imidyl ester, carboxyl, carbonamide, halogen carbonamide, sulfonyl halide, isothiocyanate, phosphoramidite, amine, azide, aryldiazo, or dithionicotine derivative.  
   
   
       43 . The oxazine derivative according to  claim 42 , wherein the at least one reactive group is an optionally substituted N-succinimidyl ester, maleimide, carboxyl, halogen acetamide, isothiocyanate, phosphoramidite, aryldiazo, azide, sulfonyl chloride, sulfo-tetrafluorphenol ester or primary or secondary amine.  
   
   
       44 . The oxazine derivative according to  claim 43 , wherein the N-succinimidyl ester is substituted with a sulfonic acid group.  
   
   
       45 . The oxazine derivative according to  claim 40 , wherein a linker compound is inserted between the at least one reactive group and a non-bridge-forming radical R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9  or R 10 .  
   
   
       46 . The oxazine derivative according to  claim 45 , wherein the linker compound comprises optionally substituted polyoxyalkyl units, optionally substituted aliphatic units, optionally substituted cycloaliphatic units or optionally substituted aromatic units.  
   
   
       47 . The oxazine derivative according to  claim 37 , wherein the salt contains a counterion selected from the group consisting of a halogen ion, a BF 4   −  ion and tetraphenyl borate.  
   
   
       48 . The oxazine derivative according to  claim 47 , wherein the counterion is a chloride ion, a bromide ion or tetraphenyl borate.  
   
   
       49 . The oxazine derivative according to  claim 37 , having the formula (II)  
     
       
         
         
             
             
         
       
     
     wherein X denotes OH, phosphoramidite or an optionally substituted N-succinimidyl ester group, or a salt thereof.  
   
   
       50 . The oxazine derivative according to  claim 37 , having the formula (III)  
     
       
         
         
             
             
         
       
     
     wherein X denotes OH, phosphoramidite or an optionally substituted N-succinimidyl ester group, or a salt thereof.  
   
   
       51 . The oxazine derivative according  claim 49 , wherein the salt thereof contains a counterion selected from the group consisting of a halogen ion, a BF 4   −  ion or tetraphenyl borate.  
   
   
       52 . The oxazine derivative according to  claim 51 , wherein the counterion is a chloride or bromide ion or tetraphenyl borate.  
   
   
       53 . The oxazine derivative according  claim 50 , wherein the salt thereof contains a counterion selected from the group consisting of a halogen ion, a BF 4   −  ion or tetraphenyl borate.  
   
   
       54 . The oxazine derivative according to  claim 53 , wherein the counterion is a chloride or bromide ion or tetraphenyl borate.  
   
   
       55 . The oxazine derivative according to  claim 37 , comprising at least one affinity marker.  
   
   
       56 . The oxazine derivative according to  claim 55 , wherein the oxazine derivative has only one affinity marker.  
   
   
       57 . The oxazine derivative according to  claim 55 , wherein the at least one affinity marker is biotin, hexa-his or hapten.  
   
   
       58 . The oxazine derivative according to  claim 40 , wherein the at least one reactive group is bound to the material to be investigated.  
   
   
       59 . The oxazine derivative according to  claim 58 , wherein the material to be investigated is i) a biological material, ii) a chemical compound, iii) a synthetic or biological micro-particle or iv) a combination thereof.  
   
   
       60 . The oxazine derivative according to  claim 59 , wherein the synthetic micro-particle is a soluble or suspendable carrier material.  
   
   
       61 . The oxazine derivative according to  claim 59 , wherein the biological micro-particle is a vesicular or virus-like particle.  
   
   
       62 . The oxazine derivative according to  claim 59 , wherein the biological material comprises at least one member selected from the group consisting of nucleotides, oligonucleotides, sugar, lipids, membranes, cells, cell constituents, DNA, RNA, peptides, proteins, antibodies, hapten and antigens.  
   
   
       63 . A kit for labelling a material to be studied, said kit comprising an oxazine derivative according to  claim 37 .  
   
   
       64 . A kit for labelling a material to be studied, said kit comprising an oxazine derivative according to  claim 49 .  
   
   
       65 . A kit for labelling a material to be studied, said kit comprising an oxazine derivative according to  claim 50 .  
   
   
       66 . A method for capturing and/or assaying a target, said method comprising contacting the target with an oxazine derivative according to  claim 37 .  
   
   
       67 . The method of  claim 66 , wherein: i) the target is a biological material or a chemical compound and the method further comprises identifying and characterizing the target; ii) the target is a biologically active substance and/or a pharmaceutically active ingredient to be located; iii) the target is an analyte and the method further comprises identifying the analyte to diagnose and/or treat a condition; iv) the target is a genomic sequence to be analyzed; or v) the target is a substrate to be purified and concentrated.  
   
   
       68 . A method for producing an oxazine derivative according to  claim 37 , wherein a first reagent having the formula VI  
     
       
         
         
             
             
         
       
     
     is reacted in an acidic medium at an elevated temperature for a time interval of 10 minutes to 5 hours with a second reagent having the formula VII  
     
       
         
         
             
             
         
       
     
   
   
       69 . The method according to  claim 68 , wherein the first reagent and the second reagent are:  
     
       
         
         
             
             
         
       
     
   
   
       70 . The method according to  claim 68 , wherein the oxazine derivative is activated by reacting with N-succinimide in a presence of N,N-dimethyl formamide and diisopropylcarbodiimide.  
   
   
       71 . A method for targeted labelling of a material to be studied, said method comprising: 
 a) preparation of a sample comprising a material to be studied;    b) reaction of the material to be studied with detection markers comprising oxazine derivatives of  claim 37  and affinity markers to provide a mixture of material to be studied, labelled with zero, one or a plurality of detection markers; and    c) separation of the material to be studied into non-labelled, singly labelled or multiply labelled fractions by affinity chromatography.    
   
   
       72 . The method according to  claim 71 , wherein the singly labelled fraction is used for a subsequent investigation of the material.  
   
   
       73 . The method according to  claim 71 , wherein the affinity markers are biotin, hexa-his or hapten.  
   
   
       74 . The method of  claim 73 , wherein each oxazine derivative has only one affinity marker.  
   
   
       75 . The method of  claim 73 , wherein each oxazine derivative has more than one affinity marker.