Hemin As A Broad-spectrum Dark Quencher For Designing Fluorescent Probes

Fluorescent probe technology is widely used in multi-dimensional real-time tracking of biomolecules,cells,tissues,and living bodies.With the advantages of noninvasion,high temporal and spatial resolution,safety,and low cost,it has become a very important tool for current biomedical investigation.Dual labeling with a fluorescent reporter and a dark quencher at either side of a cleavable substrate is a common strategy for developing such “activatable” fluorescent probes.However,for optimal performance,matching of the fluorophore and dark quencher is usually required,which limits the molecular design and complex applications.Because of the unique spectral properties,biocompatibility,easy modification,and ready availability of hemin,it can be used to simplify probe design for multicolor fluorescence analysis as a potential broad-spectrum dark quencher.Accordingly,we investigated the quenching performance of hemin in several,novel dual-labeled probes,which were designed and synthesized for fluorogenic detection of protein function under physiological and pathological conditions.The main contents are as follows:Chapter 1.The main pathways of fluorescence quenching were briefly introduced,and the progress in recent years of dark quenchers and broad-spectrum quenchers was reviewed.Then,characteristics of heme and the key enzyme system of heme metabolism in mammalian cells were briefly introduced,and the common methods of detecting the activity of heme oxygenase 1（HO-1）were summarized.Chapter 2.We herein constructed a covalently linked hemin-containing scaffold to investigate the quenching efficiency and mechanism of hemin,finding that static quenching is the cardinal mechanism of hemin to turn off a broad range of fluorescence in the visible region.On this basis,a biological thiol-activated fluorescent probe,NapSS-H,was designed and synthesized.It was unexpectedly found that fluorescence respond of Nap-SS-H could be specifically triggered on by vicinal dithiol-containing proteins（VDPs）,and the probe was further used for VDPs detection in vitro and live cells imaging.In addition,the degree of VDPs loss in oxygen-glucose deprivation model was revealed by Nap-SS-H for the first time.Chapter 3.A proof-of-concept model of E.coli lysate with HO-1 activity was constructed.It was found that the probe,RBH,mentioned in Chapter 2 could be effectively metabolized by HO-1 in this model,thereby destroying the fluorescence quenching effect of hemin and releasing the fluorescence signal.To the best of our knowledge,RBH is the first small-molecule fluorescent probe capable of determining HO-1 activity in cell lysate and imaging HO-1 in live cells.This novel probe demonstrated comparable reliability to traditional method and allowed the visual detection of different levels of HO-1 activity.