Synthesis of Thiol-Reactive,Long-Wavelength Fluorescent Phenoxazine Derivatives for Biosensor Applications

摘要

Two environmentally sensitive,long-wavelength fluorescent phenoxazine derivatives,INR and IANR,were synthesized with linkers for conjugation to the thiol group of cysteine in binding proteins.The linkers were designed based on the attachment sites at two different positions on the phenoxazine,which were chosen in order to study the orientation of the dye with respect to the binding protein.Conjugation of the dyes to the S337C maltose binding protein(MBP)mutant provided conjugates of these dyes that are capable of detecting maltose with different sensitivities.The dye INR gave a 3-fold(+200%)change in fluorescence intensity upon maltose binding when conjugated to S337C MBP with a binding constant(K_d)of 435 muM.The fluorescence change for IANR was only 20% and the K_d was 1.4 mM.Conformational analysis of the dyes by molecular modeling suggested that the linker in IANR imparted greater conformational freedom to the dye,resulting in little change in environment between the open and the closed-form conformations.The linker in INR,on the other hand,showed restricted motion,which placed the dye in different environments in the open and closed forms of the protein.Thus,design and placement of the linker play a critical role in the performance of these dyes as environmentally sensitive probes.