Coumarin-based fluorogenic agents and uses thereof for specific protein labelling

A fluorescent labelling agent comprising a compound of Formula I, or a salt thereof:

• wherein:
• R1 is OR1′ or NR2′R3′, wherein R1′, R2′ and R3′ are independently selected from hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkaryl, arylalkyl, and carboxy alkyl, alkyl being optionally substituted by hydroxyl, amino, carboxyl, carboxylic ester, amide, carbamate, or aminoalkyl;
• or R1′ and R2 or R1′ and R3 come together to form a 5, 6 or 7-membered ring which is selected from aryl, heterocyclic, heteroaryl, and heteroaromatic;
• or R2′, R2, R3′, and R3 come together independently to form at least one 5, 6 or 7-membered ring which is selected from aryl, heterocyclic, heteroaryl and heteroaromatic;
• R2, R3 and R4 are independently selected from hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkaryl, arylalkyl, carboxy alkyl, aryl, heterocyclic, heteroaryl, and heteroaromatic, alkyl being optionally substituted by hydroxyl, amino, carboxyl, carboxylic ester, amide, carbamate, or aminoalkyl;
• R5 and R6 are independently selected from hydrogen, halogen, alkyl, alkoxy, keto, carboxylate, carboxylic ester, carbamate, amide, amino, alkylamino, polyether, alkylthio, cyano, nitro, sulfonyl, X1, and X2, wherein only one of R5 and R6 is X1 or X2, and alkyl is optionally substituted by hydroxyl, amino, carboxyl, carboxylic ester, amide, carbamate, or aminoalkyl;
• X1 is
• X2 is
• and
• R7 and R8 are independently R9 or OR10, wherein R9 is selected from hydrogen, halogen, and alkyl, alkyl being optionally substituted by hydroxyl, amino, carboxyl, carboxylic ester, amide, carbamate, or aminoalkyl, and R10 is alkyl, alkyl being optionally substituted by hydroxyl, amino, carboxyl, carboxylic ester, amide, carbamate, or aminoalkyl.