Protein melt analysis using dipyrrometheneboron difluoride compounds

A method comprising:

• (a) forming a sample solution mixture comprising at least one protein and a dye of formula (I):
• wherein:
• R1 to R6 is independently selected from the group consisting of:
• hydrogen, halogen, —(CH2)nCO2H (wherein n=0 to 6), —(CH2)nCO2R (wherein n=0 to 6), cycloakyl, alkyl (1-5 carbons), aryl, heteroaryl, arylalkyl (wherein the alkyl portion is 1-5 carbon atoms), alkenyl, azido, alkynyl, and sulfo, alone or in combination,
• wherein o, m and p are independently selected from the group consisting of:
• hydrogen, halogen, —(CH2)nCO2H (wherein n=0 to 6), —(CH2)nCO2R (wherein n=0 to 6), cycloakyl, alkyl (1-5 carbons), aryl, heteroaryl, arylalkyl (wherein the alkyl portion is 1-5 carbon atoms), alkenyl, azido, alkynyl, sulfo, and a maleimidyl substituent having the structure:
• alone or in combination, with the proviso that the maleimidyl substituent occurs in formula (I) once and only once,
• wherein an alkenyl substituent is substituted or unsubstituted,
• wherein the alkenyl group is ethenyl, dienyl, or trienyl,
• wherein the one or more substituents for an alkenyl group is selected from the group consisting of:
• hydrogen, halogen, alkyl (1-5 carbon atoms), cyano, carboxylate ester, carboxamide, aryl, heteroaryl, and any combination thereof,
• wherein an aryl is selected from phenyl, 1-naphthyl, 2-naphthyl, 1-pyrenyl, 9-anthryl, pyridyl, quinolyl, and alkoxy derivatives thereof,
• wherein an aryl or heteroaryl group is substituted or unsubstituted,
• wherein the one or more substituents for an aryl or heteroaryl group is selected from the group consisting of:
• hydrogen, halogen, —(CH2)nCO2H (wherein n=0 to 6), —(CH2)nCO2R (wherein n=0 to 6) alkyl (1-5 carbons), alkoxy(wherein the alkyl portion is 1˜4 carbon atoms), and any combination thereof,
• wherein any alkyl or arylalkyl group is substituted or unsubstituted,
• wherein the one or more substituents for an alkyl or arylalkyl group is selected from the group consisting of:
• hydrogen, halogen, —(CH2)nCO2H (wherein n=0 to 6), —(CH2)nCO2R (wherein n=0 to 6), cycloakyl, alkyl (1-5 carbons), aryl, heteroaryl, arylalkyl (wherein the alkyl portion is 1-5 carbon atoms), alkenyl, azido, alkynyl, and sulfo, alone or in combination,
• wherein the alkyl group in any substituent of an aryl group may be further substituted by an ester or amide substituent, and
• wherein L is an optionally present linker and when present L is selected from alkyl (1-6 carbons), and heteroalkyl, (1-6 atoms);
• and
• (b) applying a controlled heating to the sample solution mixture;
• and
• (c) measuring fluorescence emitted over a temperature range.