Fluorescent dyes for the labeling of biological substrates

#1) Field of the Invention

#Functionalized dyes bearing moieties for the attachment to biological substrates are very important, serving in the detection of disease in biological fluids and the detection of a variety of analytes such as environmental contaminants and toxins in food.

#This invention relates to a new class of squaraine dyes which are equipped with functionalities serving in the covalent attachment of these dyes to biological substrates forming conjugates resulting in the fluorescent labeling of the substrate.

#A mixture of 3-[(N-carboxyethyl)amino]phenol, 1.48 grams and propane sulfone 2.5 g (excess) were mixed and heated in an oil bath at 125° C. for twenty minutes. The mixture dissolved in methanol and the product purified by silica gel column chromatography. The column eluted with ethyl acetate to remove bi-products and then with methanol to elute the product.

#Squaric acid 116 mg and 600 mg of 3-[(N-Carboxyethyl-N-Sulfopropyl)amino]-phenol were mixed in 10 ml of toluene and 10 ml of n-butanol in a flask and heated to reflux under an argon atmosphere with azeotropic removal of water overnight. Allowed to cool to room temperature and the product purified by silica-gel column chromatography. The solvents used to elute the column were ethyl acetate and methanol. The blue band containing the product was eluted at the end with methanol. The product characterized by ultraviolet absorption showed:

#U.V. max (methanol) 641 nm.