Development of Acedan-Based Two-Photon Fluorescent Probes for Bio-imaging Applications

Abstract

ABSTRACTPart I. Acedan-based fluorescent probes for biological applications Section 1. Two-photon mononuclear Zn(II)DPA probes for ATPAdenosine-5’-triphosphate (ATP) is a multifunctional nucleotide containing three negatively charged phosphate groups. It plays several important roles in cell biology, mainly as the “molecular unit of currency” for intracellular energy transfer in the living cells and a signaling molecule to coordinate responses to energy status, in part by modulating ion channels and activating signaling cascades. Deficiency in the ATP level can lead to various diseases such as ischemia and Parkinson’s disease. The monitoring of the ATP concentration level is thus important for the study of different cellular mechanisms, enzymatic processes, and even cell apoptosis.Available fluorescent probes for ATP use one photon microscopy (OPM) that requires short excitation wavelength which can cause unfavorable features such as shallow penetration depth, photo-bleaching, photo-damage, and cellular auto-fluorescence. These problems can be mostly alleviated by two-photon microscopy (TPM) that employs two near-infrared photons for excitation. An acedan derivative containing Zn(II)–DPA (1•Zn(II)) has been developed as a two-photon probe that selectively senses ATP (and ADP) in aqueous solution of physiological pH 7.4. The application of TP fluorescent probe was demonstrated by imaging ATP in live cells by one-photon microscopy as well as two-photon microscopy.Section 2. Acedan–based mononuclear Zn(II)DPA PPi probes over ATP Pyrophosphate (PPi) plays an important role in bioenergetics and metabolic processes such as cellular signal transduction and protein synthesis. It is also a hydrolysis product of nucleoside triphosphates such as ATP under cellular conditions. The detection of PPi has an impact on real-time DNA sequencing and also on cancer diagnosis. To study the different cellular mechanisms and enzymatic processes, monitoring of the PPi concentration is important.Our continuous efforts toward efficient fluorescent sensing systems for phosphate compounds of biological significance have led us to investigate selective pyrophosphate probes over ATP. Strategy of the designed probe is to trigger the optical change upon phosphate binding to mononuclear Zn(DPA) complex by de-coordination of the auxiliary ligand attached to zinc complex. Acedan derived mononuclear zinc-dipicolylamine complex evaluated as fluorescent sensing system for phosphate-containing anions. Among them, a zinc complex coordinated by a carboxamido group as an auxiliary ligand is found to selectively sense pyrophosphate anions over other phosphate containing anions including ATP. The relative fluorescence enhancement of the probe toward pyrophosphate over ATP is comparable to the best selectivity obtained by a fast-responding dinuclear Zn(II)-dipicolylamine complex known so far. Section 3. Reactive two-photon fluorescent probes for mercury ionsMercury is an indispensable element in the current chemical industry, yet the highly poisonous nature of mercury and its oxidized species have drawn our keen concern regarding their bioaccumulation and environmental contamination. Organic forms of mercury, typically, methylmercury (CH3HgX, X = halides, etc) are much more toxic than inorganic mercury species (HgX2). Ingestion of methylmercury contaminated fish or grain, triggered the epidemics of severe neurotoxicity which swept through Minamata Bay in Japan taking a heavy toll on its population and thus manifested the fatal threat of methylmercury to human health. Therefore, efficient detection and monitoring tools for mercury species are thus essential for assessing their contamination of the environment and living species.For the application of fluorescent probes to imaging of mercury species in cells and organs, a desirable probe is the one that enables us to monitor the probe itself and its interaction outcome with mercury ions through separate optical windows (probe–analyte dual probing). In addition to the dual probing property, securing of deep-tissue imaging is also an important concern when we apply a fluorescent probe for imaging of mercury ions in living species. A two-photon (TP) probe is promising for this purpose, as with it we can look into deeper tissue compared with the conventional one-photon-excitation probes. Furthermore, two-photon probes alleviate other unfavorable features of one-photon probes in bioimaging such as photo-bleaching, photo-damage, and cellular auto-fluorescence. Dithioacetal, DTA 1 undergoes a fast mercury ion-promoted deprotection to yield the aldehyde among the evaluated dithioacetals. The conversion accompanies a ratiometric emission change with a significant wavelength shift, enabling fluorescent imaging of both the probe and mercury ions in cells by one- and two-photon microscopy for the first time.Part II. Development of molecular probes for site-specific labeling of peptides / proteinsLabeling of protein with fluorescent molecules at specific sites in proteins allows the sensing and visualizing of protein dynamics, localization, and protein–protein interactions, is an invaluable technique to understand protein functions and networks in living cells. The genetic fusion of target proteins to fluorescent proteins (FPs) such as green fluorescent protein (GFP) provides the most specific and broadly applied labeling method. However, there are some practical limitations of this method. Site-specific labeling has significant role among labeling techniques. Most site-specific labeling reactions are done by targeting sulfhydryl groups, a very popular functional group due to its reaction specificity. Based on sulfhydryl group reactivity, maleimide and α,β-unsaturated ketones were chosen as the reacting sites for probes. Sulfhydryl group in secondary structure of α-helex reacts with maleimides and α,β-unsaturated ketones, only when they positioned at a matching distance between two reacting sites. Upon thiol addition maleimide containing probes gives ‘turn-on’ fluorescence by releasing photoinduced electron transfer (PET). Hypsochromic shift of emission results upon 1,4-addition of sulfhydryl group to α,β-unsaturated ketones, which is due to release of conjugation.Preliminary experiments from bis-maleimide support the design strategy of molecular probes developed for site-specific labeling of proteins/peptides. But these probes require elaborate in-vitro experiments to demonstrate in biological samples.