Bio-orthogonal Toolbox for Monitoring Nitric Oxide in Targeted Organelles of Live Cells and Zebrafishes

Abstract

Nitric oxide (NO), playing crucial roles as a cellular messenger and as a toxic ROS, is highly related to the physiological and pathological states of living systems. The very wide but very uneven distribution of this radical gas in the inhomogeneous biological microenvironment imposes big challenges for specifically detecting its local level in certain subcellular areas, which calls for a long list of NO probes for each target. In order to simplify the syntheses and designs of these probes, herein it is proposed to construct a versatile NO-sensing toolbox based on a bio-orthogonal concept, i.e., inverse electron demand Diels–Alder click reaction between tetrazine and strained alkyne BCN. On the one hand, rhodamine-o-phenylenediamine as the NO-responsive scaffold is coupled with a tetrazine unit to generate a general probe TMR-Tz-NO, which, to our knowledge, is the first case of the tetrazine-coupled analyte-responsive probe. On the other hand, the BCN moiety is connected to different targeting groups, such as TPP, morpholine, and Ac4ManN, targeting to mitochondria, lysosomes, and membranes, respectively. It works well to use TMR-Tz-NO to match with any targetable BCN counterpart in this toolbox to achieve the imaging of NO in the corresponding subcellular area. For example, through metabolism, Ac4ManN-BCN is effectively taken and grows on the cell membranes. The bio-orthogonal reaction between TMR-Tz-NO and Ac4ManN-BCN makes the NO probe anchored to the membrane surface permanently. The zebrafish experiment revealed that this bio-orthogonal pair can track and image the NO produced during inflammation in vivo.

Recent Development of Advanced Fluorescent Molecular Probes for Organelle-Targeted Cell Imaging

“…Because of the complex function of mitochondria, several metal ions and other reactive species, such as reactive nitrogen species (RNS) and reactive sulfur species (RSS), are involved in maintaining the activity of mitochondria [ 87 , 88 ]. Nowadays, probes for the mitochondria local imaging of ions such as Ca 2+ , Zn 2+ , Fe 2+ , Hg 2+ , RNS, such as NO, RSS, such as H 2 S, and glutathione (GSH) have been introduced; a variety of them are commercially available [ 89 , 90 , 91 , 92 , 93 , 94 ]. Based on the N-nitrosation of aromatic secondary amines, Miao et al developed a mitochondrion-targeted NO probe ( 21 , Scheme 3 ) and successfully visualized the NO levels in macrophages in the resting state or under diverse stimulations [ 95 ].…”

Bioorthogonal Chemistry in Translational Research: Advances and Opportunities

“…Zhang and Xiao developed an organelle specific NO sensing probe for theranostic applications. [54] Their approach involved incorporation of a rhodamine-ophenylenediamine NO sensing scaffold with a tetrazine unit. They further linked a BCN moiety to various organelle targeting groups, including TPP, morpholine, and Ac 4 ManN, using bioorthogonal chemistry.…”

“…Despite NO playing an essential role as a cellular messenger and generator of ROS, its improper distribution has significant implications for both physiological and pathological conditions. Zhang and Xiao developed an organelle specific NO sensing probe for theranostic applications [54] . Their approach involved incorporation of a rhodamine‐o‐phenylenediamine NO sensing scaffold with a tetrazine unit.…”

Tetrazine-Based Ratiometric Nitric Oxide Sensor Identifies Endogenous Nitric Oxide in Atherosclerosis Plaques by Riding Macrophages as a Smart Vehicle

“…Specifically, ratiometric fluorescent probes provide a quantitative mode to measure the analyte by calculating the ratio of two fluorescence signals, therefore avoiding errors caused by test instruments. Currently, plenty of excellent ratiometric fluorescent probes for detecting active small molecules have been reported. − Our group and others have reported numerous NO probes for the detection of exogenous and endogenous NO in inflammation and other pathological processes. − These probes work well at the in vitro level, with subcellular targeting ability and semiquantitating ability. However, they all lack targeting ability at the in vivo level, such as in finding the AS plaques.…”