Single step signal group-imidazole labeling of organic phosphate groups under aqueous conditions

A molecular conjugate comprising a signal group-imidazole labeling compound having the structure S--L--I, said labeling compound comprising:

• an imidazole moiety (I) wherein I is imidazole or a substituted imidazole wherein one of the three carbon atoms of I is substituted with a C.sub.1 -C.sub.6 alkyl substituent, which in turn is substituted with 0-2 of the following substituents:
• hydroxy, ether, cyano, amide, ester, vinyl, aryl, sulfate, sulfonate, halogen or sulfonamide;
• and
• a signal group S, wherein said signal group S is connected to one of the carbon atoms on the imidazole ring of said imidazole moiety I by a linking group L, and wherein when I is said substituted imidazole, said carbon atom to which L is connected is one of the two non-substituted carbon atoms of I;
• and wherein further
• the atoms of L that form a continuous chain between S and I, said atoms of L being denominated the L backbone, number no more than 12;
• the L backbone comprises at least one saturated atom when S is a boron-containing fluorescent moiety;
• and
• S and L are each devoid of sulfhydryl, primary amino, arylhydroxy and carboxyl groups, said labeling compound being covalently bound via said imidazole moiety to a free phosphate moiety on an organic biomolecule.

A method for labeling an organic substance containing a free phosphate moiety in aqueous solution comprising the steps of:

• providing a signal group-imidazole labeling compound of the formula:
• ##STR4## adding said compound to a solution containing an organic substance comprising a free phosphate moiety;
• and
• covalently linking said labeling compound via the imidazole moiety, in the presence of an aqueous carbodiimide reagent, to a free phosphate moiety on said organic substance.