Fluorescein derivative and synthetic method thereof

[0070] Example 1: Synthesis of 7'-bromofluorescein from 3-bromo-4-nitrophenol

[0071] The substrate for the synthesis of the fluorescein derivative 7'-bromofluorescein is:

[0072] -0.2 mmol 3-bromo-4-nitrophenol

[0073] -100 mM HEPES buffer solution

[0074] -50 micromolar thermostable dehalogenase (HadA G513T)

[0075] -2.0 micromolar SOD

[0076] -2.0 micromolar laccase

[0077] - Fifth FADH 2 The generation system consists of 2.0 μM FDH, 20 mM formic acid, 10.0 μM NAD + , the flavin-dependent reductase used in Example 1 is 2.0 μmol C 1 and 4.0 μmol FAD

[0078] -2 mmol D-cysteine

[0079] All solutions were mixed together in a 5 ml vessel at pH 8.0 and stirred with a magnetic bar at a temperature of 35°C. The reaction occurred when the thermostable dehalogenase solution was added to the vessel, and the reaction was allowed to complete for about 200 minutes. Then, the solution was pushed through a membrane filter (stirred cell) with a cut-off value of 10 kDa by ...

[0080] Example 2: Synthesis of 4',7'-difluorofluorescein from 2,5-difluoro-4-nitrophenol

[0081] The substrate for the synthesis of the fluorescein derivative 4',7'-difluorofluorescein is:

[0082] -1.0 mmol 2,5-difluoro-4-nitrophenol

[0083] -100 mM MOPS buffer solution

[0084] -50 micromolar HadA G513T

[0085] -2.0 micromolar SOD

[0086] -2.0 micromolar laccase

[0087] - Fourth FADH 2 Generated system consisting of 4.0 μM GDH, 20.0 mM glucose, 10.0 μM NAD + , 2.0 μM flavin-dependent reductase (HadX), 4.0 μM FAD, and 2.0 mM D-cysteine

[0088] In a pH 8.0, 5 ml vessel, mix all solutions together and stir with a magnetic bar at a temperature of 35°C. The reaction occurs when the thermostable dehalogenase solution or HadA G513T is added to the vessel and the reaction is then allowed to complete. Finally, 4',7'-difluorofluorescein, a fluorescein derivative prepared from 2,5-difluoro-4-nitrophenol, will be obtained.

[0089] Example 3: Synthesis of 4',5'-dimethylfluorescein from 2,3-dimethyl-4-nitrophenol

[0090] The substrate for the synthesis of the fluorescein derivative 4',5'-dimethylfluorescein is:

[0091] -0.5 mmol 2,3-dimethyl-4-nitrophenol

[0092] -100 mM sodium phosphate buffer solution

[0093] -100 micromolar thermostable dehalogenase (HadA G513T)

[0094] -5.0 micromolar SOD

[0095] -2.0 micromolar tyrosinase

[0096] -Second FADH 2 Generation system, including NADH and FAD, where NADH is used to generate FADH from FAD 2 A reducing agent and a group of flavin reductase substrates

[0097] -5 mmol D-cysteine

[0098] All solutions were mixed together in a 20 ml vessel at pH 7.5 and stirred with a magnetic bar at a temperature of 35°C. The reaction occurs when the thermostable dehalogenase solution or HadA G513T is added to the vessel, which is then allowed to complete. Finally, the fluorescein derivative 4',5'-dimethylfluorescein prepared from 2,3-dimethyl-4-nitrophe...