Cyanine dyes as labeling reagents for detection of biological and other materials by luminescence methods

Abstract

The present invention pertains to luminescent dyes and methods for covalently attaching the dyes to a component or mixture of components so that the components may be detected and/or quantified by luminescence detection methods. The dyes are cyanine and cyanine-type dyes that contain or are derivatized to contain a reactive group. The reactive group is covalently reactive with amine, hydroxy and/or sulfhydryl groups on the component so that the dye can be covalently bound to the component. In addition, the dyes are preferably soluble in aqueous or other medium in which the component is contained. The components to be labeled can be either biological materials, such as antibodies, antigens, peptides, nucleotides, hormones, drugs, or non-biological materials, such as polymers, glass, or other surfaces. Any luminescent or light absorbing detecting step can be employed in the method of the invention.

1 . A method for imparting luminescence to a component which contains or is derivatized to contain an amine, sulhydryl or hydroxy group in a liquid, said method comprising the steps of: 
 adding to a liquid containing said component, wherein said component is selected from the group consisting of proteins, cells, nucleic acids, sugars, carbohydrates and combinations thereof a luminescent cyanine dye having the structure                          wherein:    the dotted lines each represent carbon atoms necessary for the formation of said cyanine dye, X and Y are selected from the group consisting of O, S and CH 3 —C—CH 3 , m is an integer selected from the group consisting of 1, 2, 3 and 4, and at least one of R 1 , R 2 , R 3 , R 4  and R 7  contain a reactive group covalently reactive with amine or hydroxy groups; and    reacting said cyanine dye with said component under reaction conditions sufficient for forming a covalent bond between said reactive group and said amine or hydroxy group so that said dye labels said component.    
   
   
       2 . The method recited in  claim 1  wherein said reactive group is selected from the group consisting of isothiocyanate, isocyanate, phosphoramidite, monochlorotriazine, dichlorotriazine, mono- or di- halogen substituted pyridine, mono- or di- halogen substituted diazine, aziridine, sulfonyl halide, acid halide, hydroxysuccinimide ester, hydroxy sulfosuccinimide ester, imido ester, glyoxal and aldehyde.  
   
   
       3 . The method recited in  claim 2  wherein when any of said R 3 , R 4  and R 7  is not a reactive group, said remaining R 3 , R 4  and R 7  are selected from the group consisting of hydrogen, C 1 -C 4  alkyl or the group E-F, a polar group wherein E is a spacer group having the structure —(CH 2 ) n — and n of said spacer group is 1, 2, 3, 4 or 5, and F is hydroxy, protected hydroxy, sulfonate, sulfate, carboxyl, or lower alkyl substituted amino; and when any of R 1  and R 2  is not said reactive group, said remaining R 1  or R 2  are a C 1 -C 4  alkyl group or said E-F group and n of said spacer group is 1, 2, 3, 4 or 5.  
   
   
       4 . A luminescent cyanine dye comprising a cyanine dye having the structure  
     
       
         
         
             
             
         
       
       wherein:  
       the dotted lines each represent carbon atoms necessary for the formulation of said cyanine;  
       X and Y are independently selected from the group consisting of O, S and CH 3 —C—CH 3 ;  
       m is an integer from 1-4;  
       at least one of the groups R 1 , R 2 , R 3 , R 4  and R 7  is a reactive group, reactive with amino, sulfhydryl or hydroxy nucleophiles; and  
       when any of said R 3 , R 4  and R 7  is not a reactive group, said remaining R 3 , R 4  and R 7  are selected from the group consisting of hydrogen, C 1 -C 4  alkyl or the group E-F, a polar group wherein E is a spacer group having the structure —(CH 2 ) n — and n of said spacer group is 1, 2, 3, 4 or 5, and F is hydroxy, protected hydroxy, sulfonate, sulfate, carboxyl, or lower alkyl substituted amino; and  
       when any of R 1  and R 2  is not said reactive group, said remaining R 1  or R 2  are a C 1 -C 4  alkyl group or said E-F group and n of said spacer group is 1, 2, 3, 4 or 5.  
     
   
   
       5 . The cyanine dye recited in  claim 4  wherein said reactive group is selected from the group consisting of isothiocyanate, isocyanate, phosphoramidite, monochlorotriazine, dichlorotriazine, mono- or di-halogen substituted pyridine, mono- or di-halogen substituted diazine, aziridine, sulfonyl halide, acid halide, hydroxysuccinimide ester, hydroxy sulfosuccinimide ester, imido ester, glyoxal and aldehyde.  
   
   
       6 . A luminescent labeled component comprising: 
 a component selected from the group consisting of proteins, nucleic acids, cells, carbohydrates, sugars, and combinations thereof having amino, sulfhydryl or hydroxy groups covalently bound to a luminescent cyanine dye having the structure                          wherein:    the dotted lines each represent carbon atoms necessary for the formulation of said cyanine;    X and Y are independently selected from the group consisting of O, S and CH 3 —C—CH 3 ;    m is an integer from 1-4;    at least one of the groups R 1 , R 2 , R 3 , R 4  and R 7  is a reactive group, reactive with amino, sulfhydryl or hydroxy nucleophiles.    
   
   
       7 . The component recited in  claim 6  wherein at least one of said reactive groups is a phosphoramidite.  
   
   
       8 . The component recited in  claim 6  wherein said reactive group is selected from the group consisting of isothiocyanate, isocyanate, phosphoramidite, monochlorotriazine, dichlorotriazine, mono- or di-halogen substituted pyridine, mono- or di-halogen substituted diazine, aziridine, sulfonyl halide, acid halide, hydroxysuccinimide ester, hydroxy sulfosuccinimide ester, imido ester, glyoxal and aldehyde; and 
 when any of said R 3 , R 4  and R 7  is not a reactive group, said remaining R 3 , R 4  and R 7  are selected from the group consisting of hydrogen, C 1 -C 4  alkyl or the group E-F, a polar group wherein E is a spacer group having the structure —(CH 2 ) n — and n of said spacer group is 1, 2, 3, 4 or 5, and F is hydroxy, protected hydroxy, sulfonate, sulfate, carboxyl, or lower alkyl substituted amino; and    when any of R 1  and R 2  is not said reactive group, said remaining R 1  or R 2  are a C 1 -C 4  alkyl group or said E-F group and n of said spacer group is 1, 2, 3, 4 or 5.