Metal-Based Optical Probes for Live Cell Imaging of Nitroxyl (HNO)

Abstract

Conspectus: Nitroxyl (HNO) is a biological signaling agent that displays distinctive reactivity compared to nitric oxide (NO). As a consequence, these two reactive nitrogen species trigger different physiological responses. Selective detection of HNO over NO has been a challenge for chemists, and several fluorogenic molecular probes have been recently developed with that goal in mind. Common constructs take advantage of the HNO-induced reduction of Cu(II) to Cu(I).The sensing mechanism of such probes relies on the ability of the unpaired electron in a d orbital of the Cu(II) center to quench the fluorescence of a photoemissive ligand by either an electron or energy transfer mechanism. Experimental and theoretical mechanistic studies suggest that proton-coupled electron transfer mediates this process, and careful tuning of the copper coordination environment has led to sensors with optimized selectivity and kinetics.The current optical probes cover the visible and near-infrared regions of the spectrum. This palette of sensors comprises structurally and functionally diverse fluorophores such as coumarin 2 (blue/green emission), boron dipyrromethane (BODIPY, green emission), benzoresorufin (red emission), and dihydroxanthenes (near-infrared emission). Many of these sensors have been successfully applied to detect HNO production in live cells. For example, copper-based optical probes have been used to detect HNO production in live mammalian cells that have been treated with H 2 S and various nitrosating agents. These studies have established a link between HSNO, the smallest S-nitrosothiol, and HNO. In addition, a near-infrared HNO sensor has been used to perform multicolor/multianalyte microscopy, revealing that exogenously applied HNO elevates the concentration of intracellular mobile zinc. This mobilization of zinc ions is presumably a consequence of nitrosation of cysteine residues in zinc-chelating proteins such as metallothionein.Future challenges for the optical imaging of HNO include devising probes that can detect HNO reversibly, especially because ratiometric imaging can only report equilibrium concentrations when the sensing event is reversible. Another important aspect that needs to be addressed is the creation of probes that can sense HNO in specific subcellular locations. These tools would be useful to identify the organelles in which HNO is produced in mammalian cells and probe the intracellular signaling networks in which this reactive nitrogen species is involved. In addition, near-infrared emitting probes might be applied to detect HNO in thicker specimens, such as acute tissue slices and even live animals, enabling the investigation of the roles of HNO in physiological or pathological conditions in multicellular systems.

Mobile zinc increases rapidly in the retina after optic nerve injury and regulates ganglion cell survival and optic nerve regeneration

“…The ability of TPEN to stabilize the survival of RGCs lacking pten suggests that Zn 2+ chelation may selectively stabilize the survival of the same types of RGCs that are protected by pten deletion, although further work is needed to confirm this point. In terms of upstream mechanisms, NO produced via NOS1 mobilizes Zn 2+ from metallothioneins (102,103) in various neurological disorders (47,59), and our preliminary studies indicate that NOS1-derived NO lies upstream of Zn 2+ elevation in amacrine cell processes (104).…”

The chemical biology of HNO signaling

“…In spite of these experimental difficulties, recent studies clearly indicate that HNO possesses unique biological activity that makes it useful as a possible therapeutic agent ( vide infra ) [3,4]. Indeed, the biological effects of pharmacologically administered HNO have prompted the development of numerous HNO-donor species [5] as well as novel methods for its detection [for example, 6]. Herein will be reviewed the current understanding of the chemistry of HNO along with the targets/chemical biology that may be responsible for some of its reported biological actions and signaling.…”

Copper(ii)-doped semiconducting polymer dots for nitroxyl imaging in live cells

“…Recently, several fluorescence assays for HNO detection have been developed due to their high sensitivity, high spatiotemporal resolution, and real-time imaging ability in biological systems, including cells and tissues. 11-16 …”

“…The reported fluorescence assays for HNO detection fall into two main categories, metal-based 11, 15, 17 and phosphine-based molecular probes. 13, 18-20 However, these probes can be sensitive to fluctuations of the biological conditions, such as pH.…”

“…The reduction by HNO can lead to the disruption of the electron transfer process and thus turn on fluorescence from the Pdots. 11, 37, 38 …”