Synthesis of thiol-reactive, long-wavelength fluorescent phenoxazine derivatives for biosensor applications

Bioconjugate Chemistry
Douglas B ShermanK Joseph Thomas

Abstract

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 microM. 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 pla...Continue Reading

Citations

Jan 1, 2010·Sensors·Laurence Choulier, Karin Enander
Dec 8, 2009·Trends in Biotechnology·Galen S LovingBarbara Imperiali
Jul 14, 2016·Chemistry : a European Journal·Vincent Martinez, Maged Henary
Feb 13, 2016·Chemphyschem : a European Journal of Chemical Physics and Physical Chemistry·Irene TosiFrancesca Terenziani
Apr 4, 2017·Macromolecular Rapid Communications·Jordan C TheriotGarret M Miyake
Apr 12, 2014·Chemical Society Reviews·Zhigang YangJong Seung Kim

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