Bioorthogonal “Click Chemistry” synthesis of fluorescent derivative of Ethinyl estradiol for probing estrogen receptor on Poly(dimethylsiloxane)-Based microarrays

Abstract

To date, binding affinities of estrogen receptors (ERs) have been studied but with a struggle of achieving good biocompatibility, thermal stability, optical transparency, low manufacturing costs and robust results. Herein, we reported bioorthogonal “click chemistry” synthetic route to yield a fluorescent (Cy3) derivative of ethinyl estradiol (EE2) in order to demonstrate its importance as a probe for affinity assays of estrogen receptor (ER) on poly(dimethylsiloxane) (PDMS)-based microarrays. Furthermore, a novel three-step Mitsunobu route has been used to synthesize a 17α-alkynyl amine derivative of E2 (E2-NH2, 3) followed by its labeling with Cy3 (through EDC/NHS chemistry) in order to explore and compare its receptor binding affinity on the aforementioned PDMS platform. An indirect method was also investigated, in which ER was firstly exposed to EE2 and followed by applying of optimized bioorthogonal “click chemistry” reaction conditions against the cognate EE2 and Cy3 azide. Eventually, we found that indirect method is more specific than direct method and thus established the importance of bioorthogonal “click chemistry”. The optimized direct and indirect methods were further explored for real samples of MCF-7 and MBD-MB-231 cell lines and yielded excellent results. Conclusively, an exhibition of minimum nonspecific binding was yielded in this case which suggested that PDMS is a robust and affordable material in a common lab for developing fluorescence binding assays. Comparative docking studies have also been performed in a supporting role to reveal the interaction of Cy3-EE2 and Cy3-3 molecules with the same target of ER, present in breast cancer and which. We found a better correlation between the computed experimentally determined binding affinities as we have achieved almost equivalent or better results (RBA values) than those of earlier reports.