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Fluorescent probes to investigate nitric oxide and other reactive nitrogen species in biology (truncated form: fluorescent probes of reactive nitrogen species)
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Fluorescent probes to investigate nitric oxide and other reactive nitrogen species in biology (truncated form: fluorescent probes of reactive nitrogen species) Lindsey E McQuade and Stephen J Lippard Reactive nitrogen species (RNS), such as nitric oxide and its higher oxides, play important roles in cell signaling during many physiological and pathological events. Elucidation of the exact functions of these important biomolecules has been hampered by the inability to detect RNS reliably under biological conditions. A surge of research into RNS chemistry has resulted in the design of a new generation of fluorescent probes that are specific and sensitive for their respective RNS analytes. Progress in the field of nitric oxide, peroxynitrite, and nitroxyl sensing promises to advance our knowledge of important signaling events involving these species and lead to a better understanding of oxidative biochemistry crucial to health and disease. Address Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, United States Corresponding author: Lippard, Stephen J ([email protected])
Current Opinion in Chemical Biology 2010, 14:43–49 This review comes from a themed issue on Molecular Imaging Edited by Robert Campbell and Chris Chang Available online 16th November 2009 1367-5931/$ – see front matter # 2009 Elsevier Ltd. All rights reserved. DOI 10.1016/j.cbpa.2009.10.004
Introduction Biological transmission of a signal from an input stimulus to the appropriate physiological target is accomplished by invoking a signal transduction pathway, in which secondary messengers activate enzymes to propagate information. The signal is amplified as it is transmitted, because each wave of enzyme activation results in a greater number of proteins and messengers involved in the process. Secondary messengers are crucial to the signaling cascade and come in a variety of forms, including oxidative compounds such as reactive oxygen (ROS) and reactive nitrogen (RNS) species [1]. The prototypical RNS is nitric oxide (NO), which was identified decades ago as the endothelium derived relaxation factor that promotes vascular smooth muscle relaxation when it binds and activates soluble guanylyl cyclase [2–4]. Since then, NO has been implicated in a large array of biological functions, from normal physiological events www.sciencedirect.com
such as neurotransmission [5] and host immune system defense [6] to pathologies such as ischemia, inflammation, cancer, and neurodegeneration [1]. Moreover, as biological nitric oxide research expands it has become increasingly clear that products of NO oxidation such as nitrogen dioxide (NO2), dinitrogen trioxide (N2O3), peroxynitrite (ONOO), nitrite (NO2), nitrate (NO3), and nitroxyl (HNO) also play important roles in both physiological and pathological processes [1,7–9]. Because of the significance of these secondary messengers to human health and disease, investigating the roles of RNS continues to be an important area of research. One of the major limitations in studying these essential molecules is the lack of appropriate tools to monitor their production in vivo. Fluorescence, especially in combination with microscopy, is amenable to in vivo analyte sensing. A fluorescent probe must have certain characteristics to be useful for biological imaging. First, in order to be suitable for use under biological conditions it should be water soluble, cell-membrane permeable, non-toxic, and excitable at low-energy wavelengths that do not induce interfering autofluorescence. Second, it should display good photophysical properties such as a large difference in brightness (e f) between the on and off states. Finally, it should have excellent sensing characteristics such as selectivity, cell-trappability, reversibility, and diffusability. Moreover, it should respond rapidly and directly to the analyte of interest at its biological concentrations. Not all useful fluorescent probes share these features, but as each new generation of a probe emerges it is usually improved in one or more of the requisite properties. The present review summarizes recent progress in smallmolecule probe design and utilization in the areas of nitric oxide, peroxynitrite, and nitroxyl sensing. Although nitrite and nitrate sensing by fluorescence quenching is an active research area, the field is dominated by electrochemical and colorimetric detection methods and will not be covered here. For a more comprehensive background of RNS sensing, the interested reader is referred to additional reviews [10–15].
Small-molecule fluorescent probes for nitric oxide Because of the importance of nitric oxide in many (patho)physiological functions, development of tools to probe its presence is an active research field. Many design Current Opinion in Chemical Biology 2010, 14:43–49
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Figure 1
Organic NO-specific probes. (a) The sensing mechanism of the o-phenylenediamine type probes. The diamine molecule is quenched by a photoinduced electron transfer mechanism that is abolished upon reaction with oxidized NO and formation of a triazole species. (b) Examples of such probes using rhodamine, BODIPY, cyanine, and calcein fluorophores. (c) The sensing mechanism of the ring-opening type probes. The spirolactam is quenched because of a lack of aromaticity that is restored upon ring opening by reaction with oxidized NO.
strategies have been explored, and small molecule, nanotube [16], cellular-based [17,18], and protein-based [19] assays have been employed with great success. Two main classes have emerged in the portfolio of NO-specific small-molecule fluorescent probes: organic-based and metal-based sensors. In both instances, the goal is to alter the fluorescent properties of the probe by specific reaction with NO. Organic probes employ fluorophores with pendant functional groups that serve to quench their fluorCurrent Opinion in Chemical Biology 2010, 14:43–49
escence until restored by a specific reaction with NO. Metal-based probes take advantage of the reactivity of NO at the metal site itself or have altered NO reactivity at a site near the metal. Of the organic-based probes, the most prevalent are o-phenylenediamine constructs (Figure 1a) [20]. The electron-rich vicinal amines are thought to alter the energy of the aromatic group to facilitate photo-induced www.sciencedirect.com
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Figure 2
Metal-based NO-specific probes. (a) The sensing mechanism of CuFL. FL is quenched by a PeT mechanism from the aminoquinoline to the fluorescein. Cu(II)-binding enhances the quenching until exposure to NO, which reduces the copper and nitrosates FL to form a brightly fluorescent species. (b) MNIP-Cu, a similarly designed probe that has been applied to liver slices from A) normal mice and C) a mouse model of severe liver damage, illustrating that NO production is increased upon hepatic injury (Adapted from Ref. [29]). (c) The structure and sensing mechanism of the dimeric Ru(II) probe and its Ru-NO product.
electron transfer (PeT) and quench fluorescence. In the presence of an oxidized form of NO, an electron-poor aryl triazole is formed, which abolishes the PeT pathway and restores fluorescence. Based on this reactivity, these probes can also be considered detectors of nitrosonium ion (NO+), which exists in the cell as NO2 or N2O3. Probes incorporating fluorescein [20] were the first fluorescent sensors designed, but because of their pH sensitivity and problems with photobleaching, rhodamine-based [21] probes were developed. Subsequently, BODIPY [22,23] fluorophores were incorporated into sensing agents, and recent work exploring cyanine-based near-IR probes provides a promising option for live-tissue imaging (Figure 1b) [24]. Very recent work describes a strategy to enhance the cellular retention of sensors to increase sensitivity [25]. Increased cellular retention is speculated to increase NO sensitivity by making more intracellular sensor available for reaction and by reducing the background fluorescence such that small changes in fluorescence induced by low NO concentrations will be visible. When calcein is used as the parent fluorophore of such a probe, DCl-DA, the www.sciencedirect.com
negative charge of its two iminodiacetate groups greatly increases cellular retention in HeLa and BAEC cells. The background fluorescence is also lowered owing to reduced leakage of the triazole product out of the cell. Probes of the o-phenylenediamine scaffold have been utilized for ex vivo NO visualization. 3,4-Diamino BODIPYs have been demonstrated to be competent for cellular imaging [26]. Similarly, previously reported vicinal diaminobenzoacridine molecules react with NO and are useful for imaging purposes in Raw 264.7 macrophages [27]. A variation of the o-phenylenediamine system was recently reported in which the quenched probe is a rhodamine B spirolactam (Figure 1c) [28]. The closed ring conformation of the fluorophore abolishes aromaticity and thus eliminates fluorescence. Oxidized NO reacts with the adjacent diamines to produce the ring-opened, and therefore fluorescent, rhodamine B acylbenzotriazole, which quickly decomposes to yield rhodamine B. Organic NO probes have been useful as cellular NOdetection agents. One limitation, however, is that none of Current Opinion in Chemical Biology 2010, 14:43–49
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the organic probes discussed are reversible nor do they detect NO directly. Because metals can form direct and reversible bonds with nitric oxide, many researchers have focused on metal–ligand constructs where the ligand contains a fluorophore. Quenching of the intrinsic fluorescence, accomplished by complexation with either a paramagnetic or heavy metal, can be alleviated by reduction of the metal and/or displacement of the ligand. To date, nitric oxide sensing has been accomplished by using a wide array of metal ions, including Co(II), Fe(II), Ru(II), Rh(II), and Cu(II) [11]. The most promising of metal-based sensors for direct, cellular detection of NO is CuFL (Figure 2a) [29,30]. Upon exposure of the Cu(II)FL complex to excess nitric oxide an immediate 11-fold fluorescence enhancement occurs, which is selective for NO over NO2, NO3, H2O2, HNO, ONOO, O2, and OCl. During the reaction with NO, Cu(II) is reduced to Cu(I) and the ligand is N-nitrosated to produce the fluorescent species, FL-NO, which dissociates from the metal center. CuFL can detect endogenously produced NO from Raw 264.7 macrophages and SK-N-SH neuroblastoma; however, it freely diffuses in and out of cells. A second-generation sensor functionalized with esters was prepared that undergoes hydrolysis by intracellular esterases to yield a negatively charged, and therefore well-retained, product for use in biological studies (LM McQuade and SJ Lippard, unpublished). The sensing strategy employed in CuFL is widely applicable and has been incorporated into new NOspecific probes. A sensor was reported in which Cu(II) binding to the blue fluorophore 4-methoxy-2-(1Hnaphtho[2,3-d]imidazol-2-yl)phenol (MNIP) quenches the fluorescence (Figure 2b) [31]. Like CuFL, exposure of MNIP-Cu to excess NO results in rapid N-nitrosation of the ligand with concomitant reduction of Cu(II) to Cu(I). In addition to nitric oxide detection in cultured Raw 264.7 macrophages, MNIP-Cu was used to visualize inflammation-induced NO production in liver slices of ASHI mice, which are models for severe liver disease, versus normal Balb/c mice. Thus, CuFL and MNIP-Cu type probes are valuable for biological imaging of NO production. Copper(II) is a versatile metal for NO sensing and can be used in a wide array of platforms. Conducting metallopolymers (CMPs) are fluorescent films that can be turned into sensors by incorporation of an appropriate sensor unit. Nitrogen-containing aromatics such as bipyridine and terpyridine groups that bind Cu(II) have been integrated into CMPs as NO-sensitive reaction sites [32–34]. The paramagnetic Cu(II) quenches the intrinsic film fluorescence, but reaction of NO with Cu(II) to produce the Cu(I)-polymer film results in restoration of the film emission. The most recent report of these type of sensors utilizes a cationic poly(fluorene) polymer containing 10% Current Opinion in Chemical Biology 2010, 14:43–49
bis(imidazole) incorporation into its backbone as the Cu(II) chelate [35]. Addition of Cu(II) caused an 80% reduction of polymer fluorescence that was restored upon reaction with NO. There are a few new sensor designs of note that do not contain Cu(II) or result in N-nitrosation of a ligand. One example is a dual-platform system that utilizes the rapid reaction of NO with deoxymyoglobin (Mb(Fe2+)) to modulate the fluorescence of an appended stilbene fluorophore, 4-acetamido-40 -isothiocyanoatostilbene-2,20 -disulfonic acid) (STIS) [36]. The heme quenches the stilbene by an energy transfer mechanism that is relieved upon formation of the Mb(Fe2+)-STIS-NO complex. The authors used this system to measure NO production in rabbit trachea. Another design utilizes dimeric Ru(II) complexes ligated by the fluorescent ligands dmpzn and dmpza (Figure 2c) [37]. The heavy ruthenium atoms quench the ligand fluorescence, but bubbling NO through a methanolic solution of the sensor results in a 2–3-fold increase in fluorescence. The fluorophore remains ligated to the metal, and fluorescence induction is suggested to result from the attenuated interaction of the ligand with the heavy metal center after NO binds.
Small-molecule fluorescent probes for peroxynitrite Increased understanding of the role of peroxynitrite as a mediator of the immune response against pathogens and as a player in many pathogenic processes has spurred an interest in developing probes for its detection in vivo [9]. Peroxynitrite sensors are typically organic molecules, usually a quenched fluorescent dye, that react with ONOO to produce a highly fluorescent oxidized species. Examples such as 2,7-dichlorodihydrofluorescein diacetate (DCFH2) and dihydrorhodamine 123 (RhH2) have reduced xanthene rings that are oxidized in the presence of peroxynitrite to form fully conjugated, and therefore fluorescent, dichlorofluorescein (DCF) and rhodamine 123 (Rh), respectively. These oxidations are not specific to peroxynitrite, however, and can be accomplished by OH, NO2, CO3, Fe(II), Fe(III)/ascorbate, Fe(III)/ EDTA, cytochrome c, HOCl, and H2O2 in the presence of peroxidases in the case of RhH2 [10,13,14]. Oxidation of biomolecules specifically by peroxynitrite has been accomplished in the case of folic acid, where ONOO oxidizes the hydroxyl group of the pterin moiety to produce a brightly fluorescent oxidized species [38]. The trend in newer generation ONOO probes has been to incorporate a reactive moiety in the fluorophore that undergoes a specific reaction with peroxynitrite. These sensors elicit turn-on fluorescence, so the reactive moiety quenches the fluorophore by a PeT mechanism until it is oxidized by ONOO, much like the organic NO sensors www.sciencedirect.com
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Figure 3
Peroxynitrite-specific probes. (a) The sensing mechanism of the aryloxy-amine type probes. The aryloxy-amine quenches the fluorophore by a PeT mechanism that is abolished upon reaction with highly reactive oxygen species and liberation of p-benzoquinoneimine. b) The sensing mechanism of the activated ketone type probes. The activated ketone reacts specifically with ONOO to form an unquenched dieneone product that does not require cleavage of an aryl-oxy bond for fluorescence restoration.
based on the o-phenylenediamine scaffold. In this manner fluorescein-based [39], rhodamine-based [40], and calcein-based [25] probes were designed to detect highly reactive oxygen species (hROS) such as hydroxyl radicals (OH), hypochlorite (OCl), and peroxynitrite using aryloxy-phenols or aryloxy-amines as the hROS responsive moiety (Figure 3a). Upon reaction with hROS the fluorescent species is recovered and 1,4-benzoquinone or p-benzoquinoneimine is produced. This design, however, is not specific for peroxynitrite. For example, the calcein derivative APC exhibits a 33-fold fluorescence enhancement when exposed to OH, a 19-fold enhancement with ONOO, and a 640-fold enhancement with OCl. The fluorescein-based and rhodamine-based probes also follow this pattern of selectivity. To achieve specificity, probes were designed with an activated ketone appended to one of the phenolic oxygens of the fluorescein xanthene ring [41]. Peroxynitrite selectively reacts with the ketone to form a dioxirane intermediate, which rapidly oxidizes the phenyl group to produce a new ketone and release the fluorophore to restore fluorescence. Recently, a similar BODIPY probe, HKGreen-2, was prepared that does not require cleavage of the oxidized phenyl group to restore fluorescence, a feature of the first generation probe that limited its effectiveness (Figure 3b) [42]. The dieneone product is sufficiently different in energy from the initial ketone to abolish the PeT pathway www.sciencedirect.com
and restore fluorescence. HKGreen-2 is selective for ONOO over other biologically relevant ROS and RNS and produces a 69-fold fluorescent enhancement in the presence of excess peroxynitrite. Ex vivo experiments using J774.1 murine macrophages co-incubated with the RNS/ROS producing combination of lypopolysaccharide/interferon-g (LPS/IFN-g) and phorbol myristate acetate (PMA) as well as HKGreen-2 indicate that the probe can selectively detect iNOS-induced ONOO formation in live cells.
Small-molecule fluorescent probes for nitroxyl Nitroxyl remains less well studied in comparison to other RNS, although it has been implicated in many (patho)physiologies such as increasing cardiac output, protecting or exacerbating ischemia-reperfusion toxicity, and modifying thiol activity [7]. One of the major challenges in nitroxyl probe design is discriminating nitroxyl from nitric oxide, and as such very few HNO-specific optical probes exist. The most effective strategy thus far is to use metal-based sensing platforms to distinguish the reactivity of HNO from NO at the metal center. Manganese(III) meso-(tetrakis(4-sulfonato-phenyl)) porphyrinate (MnIIITPPS) selectively undergoes reductive nitrosylation with HNO-donors but not NO-donors to form Mn(II)-NO complexes [43]. Formation of a Mn(II)-NO from the Mn(III) Current Opinion in Chemical Biology 2010, 14:43–49
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species caused a large change in absorbance; however, the complex is unstable in the presence of O2 and is quickly oxidized to the initial Mn(III) species. This instability makes metal porphyrins inadequate for quantitative biological HNO sensing. Recently, MnIIITPPS was incorporated into an aminoalkoxysilane-derived xerogel decorated with trimethoxysilyl-terminated poly(amidoamine-organosilicon) dendrimers that is NO-permeable but poorly O2-permeable to produce quantitative, HNO-responsive films [44]. Unfortunately, HNO dimerization at high nitroxyl concentrations and slow HNO complex formation within the film limit this probe in its current state.
Acknowledgements We thank the National Science Foundation (CHE-061194) for support of research from our laboratory that was covered in this review.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
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Fluorescence-based nitroxyl sensing has been accomplished using conducting metallopolymers. Previous work produced NO-sensing CMPs containing copper(II)-binding oligopyridyl units incorporated into poly( p-phenyleneethynylene), poly( p-phenylenevinylene), poly(thiophene), and poly(fluorene) derivatives [32–34]. The CP complexes are quenched by the paramagnetic Cu(II), and the Cu(II)-CP complexes formed react with NO to reduce Cu(II) to Cu(I), thereby producing a fluorescent Cu(I)-CP species. These polymers exhibit competitive binding of Cu(II) between the intended metal-chelating bipyridyl units and the water-solubilizing side chains. When the bipyridyl units are replaced with bithiophene units the binding of Cu(II) to the CMP main chain is reduced [45]. The quenched Cu(II)-polymer becomes fluorescent upon exposure to excess NO in unbuffered solutions, but under simulated biological conditions results in a decrease in emission due to precipitation of the probe. When the same CMP is exposed to excess Angeli’s salt the fluorescence increases by 2.1-fold, making the polymer a selective HNO probe under simulated biological conditions.
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Conclusions and future directions
13. Wardman P: Fluorescent and luminescent probes for measurement of oxidative and nitrosative species in cells and tissues: progress, pitfalls, and prospects. Free Radic Biol Med 2007, 43:995-1022.
The field of reactive nitrogen species sensing is an active and exciting area of research promoted by the increased interest into the role of RNS as signaling agents in biology. The NO-sensing domain has been improved by increasing the biocompatibility of probes such that tissue and animal experiments can now be considered. Peroxynitrite detection has been made possible by designing probes that react specifically with ONOO, instead of the standard ‘global-oxidation’ responsive sensors. A few metal-based systems that can discriminate HNO from NO have been described, allowing the field of nitroxyl detection to emerge. The major design feature that needs to be addressed is reversibility, which is required to get true spatiotemporal resolution. In addition, subcellular localization and ratiometric RNS detection, both of which have been achieved in cation sensing, would be valuable for determining signal localization and quantification, respectively. Current Opinion in Chemical Biology 2010, 14:43–49
10. Gomes A, Fernandes E, Lima J: Use of fluorescence probes for detection of reactive nitrogen species: a review. J Fluoresc 2006, 16:119-139. 11. Lim MH, Lippard SJ: Metal-based turn-on fluorescent probes for sensing nitric oxide. Acc Chem Res 2007, 40:41-51. 12. Nagano T, Yoshimura T: Bioimaging of nitric oxide. Chem Rev 2002, 102:1235-1269.
14. Wardman P: Methods to measure the reactivity of peroxynitrite-derived oxidants toward reduced fluoresceins and rhodamines.Nitric Oxide, Pt G: Oxidative and Nitrosative Stress in Redox Regulation of Cell Signaling. 2008: pp. 261–282. 15. Hetrick EM, Schoenfisch MH: Analytical chemistry of nitric oxide. Annu Rev Anal Chem 2009, 2:409-433. 16. Kim J-H, Heller DA, Jin H, Barone PW, Song C, Zhang J, Trudel LJ, Wogan GN, Tannenbaum SR, Strano MS: The rational design of nitric oxide selectivity in single-walled carbon nanotube nearinfrared fluorescence sensors for biological detection. Nat Chem 2009, 1:473-481. 17. Sato M, Hida N, Umezawa Y: Imaging the nanomolar range of nitric oxide with an amplifier-coupled fluorescent indicator in living cells. Proc Natl Acad Sci U S A 2005, 102:14515-14520. 18. Sato M, Nakajima T, Goto M, Umezawa Y: Cell-based indicator to visualize picomolar dynamics of nitric oxide release from living cells. Anal Chem 2006, 78:8175-8182. www.sciencedirect.com
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19. Boon EM, Marletta MA: Sensitive and selective detection of nitric oxide using an H-NOX domain. J Am Chem Soc 2006, 128:10022-10023.
This paper expands on the Cu(II)-based direct NO-sensing strategy by constructing a blue fluorescent probe that can image NO in hepatic tissue slices.
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32. Do L, Smith RC, Tennyson AG, Lippard SJ: Luminescent properties of water-soluble conjugated metallopolymers and their application to fluorescent nitric oxide detection. Inorg Chem 2006, 45:8998-9005.
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33. Smith RC, Tennyson AG, Lim MH, Lippard SJ: Conjugated polymer-based fluorescence turn-on sensor for nitric oxide. Org Lett 2005, 7:3573-3575.
22. Gabe Y, Ueno T, Urano Y, Kojima H, Nagano T: Tunable design strategy for fluorescence probes based on 4-substituted BODIPY chromophore: improvement of highly sensitive fluorescence probe for nitric oxide. Anal Bioanal Chem 2006, 386:621-626. 23. Gabe Y, Urano Y, Kikuchi K, Kojima H, Nagano T: Highly sensitive fluorescence probes for nitric oxide based on boron dipyrromethene chromophore-rational design of potentially useful bioimaging fluorescence probe. J Am Chem Soc 2004, 126:3357-3367. 24. Sasaki E, Kojima H, Nishimatsu H, Urano Y, Kikuchi K, Hirata Y, Nagano T: Highly sensitive near-infrared fluorescent probes for nitric oxide and their application to isolated organs. J Am Chem Soc 2005, 127:3684-3685. 25. Izumi S, Urano Y, Hanaoka K, Terai T, Nagano T: A simple and effective strategy to increase the sensitivity of fluorescence probes in living cells. J Am Chem Soc 2009, 131:10189-10200. This paper employs calcein as the fluorophore for a diamine-to-triazole type NO probe and an aryloxy-amine type hROS type probe. The calcein increases cellular retention, which in turn increases sensitivity. 26. Huang KJ, Wang H, Ma M, Zhang X, Zhang HS: Real-time imaging of nitric oxide production in living cells with 1,3,5,7tetramethyl-2,6-dicarbethoxy-8-(30 ,40 diaminophenyl)difluoroboradiaza-s-indacence by invert fluorescence microscope. Nitric Oxide 2007, 16:36-43. 27. Hu JX, Yin LL, Xu KH, Gao JJ, Tong LL, Tang B: Vicinal diaminobenzoacridine used as the fluorescent probe for trace nitric oxide determination by flow injection spectrofluorimetry and macrophage cells imaging. Anal Chim Acta 2008, 606:57-62. 28. Zheng H, Shang GO, Yang SY, Gao X, Xu JG: Fluorogenic and chromogenic rhodamine spirolactam based probe for nitric oxide by spiro ring opening reaction. Org Lett 2008, 10:2357-2360. 29. Lim MH, Wong BA, Pitcock WH, Mokshagundam D, Baik MH, Lippard SJ: Direct nitric oxide detection in aqueous solution by copper(II) fluorescein complexes. J Am Chem Soc 2006, 128:14364-14373.
34. Smith RC, Tennyson AG, Won AC, Lippard SJ: Conjugated metallopolymers for fluorescent turn-on detection of nitric oxide. Inorg Chem 2006, 45:9367-9373. 35. Xing CF, Yu MH, Wang S, Shi ZQ, Li YH, Zhu DB: Fluorescence turn-on detection of nitric oxide in aqueous solution using cationic conjugated polyelectrolytes. Macromol Rapid Commun 2007, 28:241-245. 36. Chen O, Uzaner N, Priel Z, Likhtenshtein GI: Novel fluorescence method for real-time monitoring of nitric oxide dynamics in nanoscale concentration. J Biochem Biophys Methods 2008, 70:1006-1013. 37. Ortiz M, Torrens M, Mola JL, Ortiz PJ, Fragoso A, Diaz A, Cao R, Prados P, de Mendoza J, Otero A et al.: Nitric oxide binding and photodelivery based on ruthenium(II) complexes of 4-arylazo3,5-dimethylpyrazole. Dalton Trans 2008:3559-3566. 38. Huang JC, Li DJ, Diao JC, Hou J, Yuan JL, Zou GL: A novel fluorescent method for determination of peroxynitrite using folic acid as a probe. Talanta 2007, 72:1283-1287. 39. Setsukinai K, Urano Y, Kakinuma K, Majima HJ, Nagano T: Development of novel fluorescence probes that can reliably detect reactive oxygen species and distinguish specific species. J Biol Chem 2003, 278:3170-3175. 40. Koide Y, Urano Y, Kenmoku S, Kojima H, Nagano T: Design and synthesis of fluorescent probes for selective detection of highly reactive oxygen species in mitochondria of living cells. J Am Chem Soc 2007, 129:10324-10325. 41. Yang D, Wang HL, Sun ZN, Chung NW, Shen JG: A highly selective fluorescent probe for the detection and imaging of peroxynitrite in living cells. J Am Chem Soc 2006, 128:6004-6005. 42. Sun ZN, Wang HL, Liu FQ, Chen Y, Tam PKH, Yang D: BODIPY based fluorescent probe for peroxynitrite detection and imaging in living cells. Org Lett 2009, 11:1887-1890. This letter presents a highly specific peroxynitrite probe with enough sensitivity to image endogenously produced peroxynitrite in live cells. 43. Marti MA, Bari SE, Estrin DA, Doctorovich F: Discrimination of nitroxyl and nitric oxide by water-soluble Mn(III) porphyrins. J Am Chem Soc 2005, 127:4680-4684.
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Current Opinion in Chemical Biology 2010, 14:43–49
Fluorescent probes to investigate nitric oxide and other reactive nitrogen species in biology (truncated form: fluorescent probes of reactive nitrogen species) Lindsey E McQuade and Stephen J Lippard Reactive nitrogen species (RNS), such as nitric oxide and its higher oxides, play important roles in cell signaling during many physiological and pathological events. Elucidation of the exact functions of these important biomolecules has been hampered by the inability to detect RNS reliably under biological conditions. A surge of research into RNS chemistry has resulted in the design of a new generation of fluorescent probes that are specific and sensitive for their respective RNS analytes. Progress in the field of nitric oxide, peroxynitrite, and nitroxyl sensing promises to advance our knowledge of important signaling events involving these species and lead to a better understanding of oxidative biochemistry crucial to health and disease. Address Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, United States Corresponding author: Lippard, Stephen J ([email protected])
Current Opinion in Chemical Biology 2010, 14:43–49 This review comes from a themed issue on Molecular Imaging Edited by Robert Campbell and Chris Chang Available online 16th November 2009 1367-5931/$ – see front matter # 2009 Elsevier Ltd. All rights reserved. DOI 10.1016/j.cbpa.2009.10.004
Introduction Biological transmission of a signal from an input stimulus to the appropriate physiological target is accomplished by invoking a signal transduction pathway, in which secondary messengers activate enzymes to propagate information. The signal is amplified as it is transmitted, because each wave of enzyme activation results in a greater number of proteins and messengers involved in the process. Secondary messengers are crucial to the signaling cascade and come in a variety of forms, including oxidative compounds such as reactive oxygen (ROS) and reactive nitrogen (RNS) species [1]. The prototypical RNS is nitric oxide (NO), which was identified decades ago as the endothelium derived relaxation factor that promotes vascular smooth muscle relaxation when it binds and activates soluble guanylyl cyclase [2–4]. Since then, NO has been implicated in a large array of biological functions, from normal physiological events www.sciencedirect.com
such as neurotransmission [5] and host immune system defense [6] to pathologies such as ischemia, inflammation, cancer, and neurodegeneration [1]. Moreover, as biological nitric oxide research expands it has become increasingly clear that products of NO oxidation such as nitrogen dioxide (NO2), dinitrogen trioxide (N2O3), peroxynitrite (ONOO), nitrite (NO2), nitrate (NO3), and nitroxyl (HNO) also play important roles in both physiological and pathological processes [1,7–9]. Because of the significance of these secondary messengers to human health and disease, investigating the roles of RNS continues to be an important area of research. One of the major limitations in studying these essential molecules is the lack of appropriate tools to monitor their production in vivo. Fluorescence, especially in combination with microscopy, is amenable to in vivo analyte sensing. A fluorescent probe must have certain characteristics to be useful for biological imaging. First, in order to be suitable for use under biological conditions it should be water soluble, cell-membrane permeable, non-toxic, and excitable at low-energy wavelengths that do not induce interfering autofluorescence. Second, it should display good photophysical properties such as a large difference in brightness (e f) between the on and off states. Finally, it should have excellent sensing characteristics such as selectivity, cell-trappability, reversibility, and diffusability. Moreover, it should respond rapidly and directly to the analyte of interest at its biological concentrations. Not all useful fluorescent probes share these features, but as each new generation of a probe emerges it is usually improved in one or more of the requisite properties. The present review summarizes recent progress in smallmolecule probe design and utilization in the areas of nitric oxide, peroxynitrite, and nitroxyl sensing. Although nitrite and nitrate sensing by fluorescence quenching is an active research area, the field is dominated by electrochemical and colorimetric detection methods and will not be covered here. For a more comprehensive background of RNS sensing, the interested reader is referred to additional reviews [10–15].
Small-molecule fluorescent probes for nitric oxide Because of the importance of nitric oxide in many (patho)physiological functions, development of tools to probe its presence is an active research field. Many design Current Opinion in Chemical Biology 2010, 14:43–49
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Figure 1
Organic NO-specific probes. (a) The sensing mechanism of the o-phenylenediamine type probes. The diamine molecule is quenched by a photoinduced electron transfer mechanism that is abolished upon reaction with oxidized NO and formation of a triazole species. (b) Examples of such probes using rhodamine, BODIPY, cyanine, and calcein fluorophores. (c) The sensing mechanism of the ring-opening type probes. The spirolactam is quenched because of a lack of aromaticity that is restored upon ring opening by reaction with oxidized NO.
strategies have been explored, and small molecule, nanotube [16], cellular-based [17,18], and protein-based [19] assays have been employed with great success. Two main classes have emerged in the portfolio of NO-specific small-molecule fluorescent probes: organic-based and metal-based sensors. In both instances, the goal is to alter the fluorescent properties of the probe by specific reaction with NO. Organic probes employ fluorophores with pendant functional groups that serve to quench their fluorCurrent Opinion in Chemical Biology 2010, 14:43–49
escence until restored by a specific reaction with NO. Metal-based probes take advantage of the reactivity of NO at the metal site itself or have altered NO reactivity at a site near the metal. Of the organic-based probes, the most prevalent are o-phenylenediamine constructs (Figure 1a) [20]. The electron-rich vicinal amines are thought to alter the energy of the aromatic group to facilitate photo-induced www.sciencedirect.com
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Figure 2
Metal-based NO-specific probes. (a) The sensing mechanism of CuFL. FL is quenched by a PeT mechanism from the aminoquinoline to the fluorescein. Cu(II)-binding enhances the quenching until exposure to NO, which reduces the copper and nitrosates FL to form a brightly fluorescent species. (b) MNIP-Cu, a similarly designed probe that has been applied to liver slices from A) normal mice and C) a mouse model of severe liver damage, illustrating that NO production is increased upon hepatic injury (Adapted from Ref. [29]). (c) The structure and sensing mechanism of the dimeric Ru(II) probe and its Ru-NO product.
electron transfer (PeT) and quench fluorescence. In the presence of an oxidized form of NO, an electron-poor aryl triazole is formed, which abolishes the PeT pathway and restores fluorescence. Based on this reactivity, these probes can also be considered detectors of nitrosonium ion (NO+), which exists in the cell as NO2 or N2O3. Probes incorporating fluorescein [20] were the first fluorescent sensors designed, but because of their pH sensitivity and problems with photobleaching, rhodamine-based [21] probes were developed. Subsequently, BODIPY [22,23] fluorophores were incorporated into sensing agents, and recent work exploring cyanine-based near-IR probes provides a promising option for live-tissue imaging (Figure 1b) [24]. Very recent work describes a strategy to enhance the cellular retention of sensors to increase sensitivity [25]. Increased cellular retention is speculated to increase NO sensitivity by making more intracellular sensor available for reaction and by reducing the background fluorescence such that small changes in fluorescence induced by low NO concentrations will be visible. When calcein is used as the parent fluorophore of such a probe, DCl-DA, the www.sciencedirect.com
negative charge of its two iminodiacetate groups greatly increases cellular retention in HeLa and BAEC cells. The background fluorescence is also lowered owing to reduced leakage of the triazole product out of the cell. Probes of the o-phenylenediamine scaffold have been utilized for ex vivo NO visualization. 3,4-Diamino BODIPYs have been demonstrated to be competent for cellular imaging [26]. Similarly, previously reported vicinal diaminobenzoacridine molecules react with NO and are useful for imaging purposes in Raw 264.7 macrophages [27]. A variation of the o-phenylenediamine system was recently reported in which the quenched probe is a rhodamine B spirolactam (Figure 1c) [28]. The closed ring conformation of the fluorophore abolishes aromaticity and thus eliminates fluorescence. Oxidized NO reacts with the adjacent diamines to produce the ring-opened, and therefore fluorescent, rhodamine B acylbenzotriazole, which quickly decomposes to yield rhodamine B. Organic NO probes have been useful as cellular NOdetection agents. One limitation, however, is that none of Current Opinion in Chemical Biology 2010, 14:43–49
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the organic probes discussed are reversible nor do they detect NO directly. Because metals can form direct and reversible bonds with nitric oxide, many researchers have focused on metal–ligand constructs where the ligand contains a fluorophore. Quenching of the intrinsic fluorescence, accomplished by complexation with either a paramagnetic or heavy metal, can be alleviated by reduction of the metal and/or displacement of the ligand. To date, nitric oxide sensing has been accomplished by using a wide array of metal ions, including Co(II), Fe(II), Ru(II), Rh(II), and Cu(II) [11]. The most promising of metal-based sensors for direct, cellular detection of NO is CuFL (Figure 2a) [29,30]. Upon exposure of the Cu(II)FL complex to excess nitric oxide an immediate 11-fold fluorescence enhancement occurs, which is selective for NO over NO2, NO3, H2O2, HNO, ONOO, O2, and OCl. During the reaction with NO, Cu(II) is reduced to Cu(I) and the ligand is N-nitrosated to produce the fluorescent species, FL-NO, which dissociates from the metal center. CuFL can detect endogenously produced NO from Raw 264.7 macrophages and SK-N-SH neuroblastoma; however, it freely diffuses in and out of cells. A second-generation sensor functionalized with esters was prepared that undergoes hydrolysis by intracellular esterases to yield a negatively charged, and therefore well-retained, product for use in biological studies (LM McQuade and SJ Lippard, unpublished). The sensing strategy employed in CuFL is widely applicable and has been incorporated into new NOspecific probes. A sensor was reported in which Cu(II) binding to the blue fluorophore 4-methoxy-2-(1Hnaphtho[2,3-d]imidazol-2-yl)phenol (MNIP) quenches the fluorescence (Figure 2b) [31]. Like CuFL, exposure of MNIP-Cu to excess NO results in rapid N-nitrosation of the ligand with concomitant reduction of Cu(II) to Cu(I). In addition to nitric oxide detection in cultured Raw 264.7 macrophages, MNIP-Cu was used to visualize inflammation-induced NO production in liver slices of ASHI mice, which are models for severe liver disease, versus normal Balb/c mice. Thus, CuFL and MNIP-Cu type probes are valuable for biological imaging of NO production. Copper(II) is a versatile metal for NO sensing and can be used in a wide array of platforms. Conducting metallopolymers (CMPs) are fluorescent films that can be turned into sensors by incorporation of an appropriate sensor unit. Nitrogen-containing aromatics such as bipyridine and terpyridine groups that bind Cu(II) have been integrated into CMPs as NO-sensitive reaction sites [32–34]. The paramagnetic Cu(II) quenches the intrinsic film fluorescence, but reaction of NO with Cu(II) to produce the Cu(I)-polymer film results in restoration of the film emission. The most recent report of these type of sensors utilizes a cationic poly(fluorene) polymer containing 10% Current Opinion in Chemical Biology 2010, 14:43–49
bis(imidazole) incorporation into its backbone as the Cu(II) chelate [35]. Addition of Cu(II) caused an 80% reduction of polymer fluorescence that was restored upon reaction with NO. There are a few new sensor designs of note that do not contain Cu(II) or result in N-nitrosation of a ligand. One example is a dual-platform system that utilizes the rapid reaction of NO with deoxymyoglobin (Mb(Fe2+)) to modulate the fluorescence of an appended stilbene fluorophore, 4-acetamido-40 -isothiocyanoatostilbene-2,20 -disulfonic acid) (STIS) [36]. The heme quenches the stilbene by an energy transfer mechanism that is relieved upon formation of the Mb(Fe2+)-STIS-NO complex. The authors used this system to measure NO production in rabbit trachea. Another design utilizes dimeric Ru(II) complexes ligated by the fluorescent ligands dmpzn and dmpza (Figure 2c) [37]. The heavy ruthenium atoms quench the ligand fluorescence, but bubbling NO through a methanolic solution of the sensor results in a 2–3-fold increase in fluorescence. The fluorophore remains ligated to the metal, and fluorescence induction is suggested to result from the attenuated interaction of the ligand with the heavy metal center after NO binds.
Small-molecule fluorescent probes for peroxynitrite Increased understanding of the role of peroxynitrite as a mediator of the immune response against pathogens and as a player in many pathogenic processes has spurred an interest in developing probes for its detection in vivo [9]. Peroxynitrite sensors are typically organic molecules, usually a quenched fluorescent dye, that react with ONOO to produce a highly fluorescent oxidized species. Examples such as 2,7-dichlorodihydrofluorescein diacetate (DCFH2) and dihydrorhodamine 123 (RhH2) have reduced xanthene rings that are oxidized in the presence of peroxynitrite to form fully conjugated, and therefore fluorescent, dichlorofluorescein (DCF) and rhodamine 123 (Rh), respectively. These oxidations are not specific to peroxynitrite, however, and can be accomplished by OH, NO2, CO3, Fe(II), Fe(III)/ascorbate, Fe(III)/ EDTA, cytochrome c, HOCl, and H2O2 in the presence of peroxidases in the case of RhH2 [10,13,14]. Oxidation of biomolecules specifically by peroxynitrite has been accomplished in the case of folic acid, where ONOO oxidizes the hydroxyl group of the pterin moiety to produce a brightly fluorescent oxidized species [38]. The trend in newer generation ONOO probes has been to incorporate a reactive moiety in the fluorophore that undergoes a specific reaction with peroxynitrite. These sensors elicit turn-on fluorescence, so the reactive moiety quenches the fluorophore by a PeT mechanism until it is oxidized by ONOO, much like the organic NO sensors www.sciencedirect.com
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Figure 3
Peroxynitrite-specific probes. (a) The sensing mechanism of the aryloxy-amine type probes. The aryloxy-amine quenches the fluorophore by a PeT mechanism that is abolished upon reaction with highly reactive oxygen species and liberation of p-benzoquinoneimine. b) The sensing mechanism of the activated ketone type probes. The activated ketone reacts specifically with ONOO to form an unquenched dieneone product that does not require cleavage of an aryl-oxy bond for fluorescence restoration.
based on the o-phenylenediamine scaffold. In this manner fluorescein-based [39], rhodamine-based [40], and calcein-based [25] probes were designed to detect highly reactive oxygen species (hROS) such as hydroxyl radicals (OH), hypochlorite (OCl), and peroxynitrite using aryloxy-phenols or aryloxy-amines as the hROS responsive moiety (Figure 3a). Upon reaction with hROS the fluorescent species is recovered and 1,4-benzoquinone or p-benzoquinoneimine is produced. This design, however, is not specific for peroxynitrite. For example, the calcein derivative APC exhibits a 33-fold fluorescence enhancement when exposed to OH, a 19-fold enhancement with ONOO, and a 640-fold enhancement with OCl. The fluorescein-based and rhodamine-based probes also follow this pattern of selectivity. To achieve specificity, probes were designed with an activated ketone appended to one of the phenolic oxygens of the fluorescein xanthene ring [41]. Peroxynitrite selectively reacts with the ketone to form a dioxirane intermediate, which rapidly oxidizes the phenyl group to produce a new ketone and release the fluorophore to restore fluorescence. Recently, a similar BODIPY probe, HKGreen-2, was prepared that does not require cleavage of the oxidized phenyl group to restore fluorescence, a feature of the first generation probe that limited its effectiveness (Figure 3b) [42]. The dieneone product is sufficiently different in energy from the initial ketone to abolish the PeT pathway www.sciencedirect.com
and restore fluorescence. HKGreen-2 is selective for ONOO over other biologically relevant ROS and RNS and produces a 69-fold fluorescent enhancement in the presence of excess peroxynitrite. Ex vivo experiments using J774.1 murine macrophages co-incubated with the RNS/ROS producing combination of lypopolysaccharide/interferon-g (LPS/IFN-g) and phorbol myristate acetate (PMA) as well as HKGreen-2 indicate that the probe can selectively detect iNOS-induced ONOO formation in live cells.
Small-molecule fluorescent probes for nitroxyl Nitroxyl remains less well studied in comparison to other RNS, although it has been implicated in many (patho)physiologies such as increasing cardiac output, protecting or exacerbating ischemia-reperfusion toxicity, and modifying thiol activity [7]. One of the major challenges in nitroxyl probe design is discriminating nitroxyl from nitric oxide, and as such very few HNO-specific optical probes exist. The most effective strategy thus far is to use metal-based sensing platforms to distinguish the reactivity of HNO from NO at the metal center. Manganese(III) meso-(tetrakis(4-sulfonato-phenyl)) porphyrinate (MnIIITPPS) selectively undergoes reductive nitrosylation with HNO-donors but not NO-donors to form Mn(II)-NO complexes [43]. Formation of a Mn(II)-NO from the Mn(III) Current Opinion in Chemical Biology 2010, 14:43–49
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species caused a large change in absorbance; however, the complex is unstable in the presence of O2 and is quickly oxidized to the initial Mn(III) species. This instability makes metal porphyrins inadequate for quantitative biological HNO sensing. Recently, MnIIITPPS was incorporated into an aminoalkoxysilane-derived xerogel decorated with trimethoxysilyl-terminated poly(amidoamine-organosilicon) dendrimers that is NO-permeable but poorly O2-permeable to produce quantitative, HNO-responsive films [44]. Unfortunately, HNO dimerization at high nitroxyl concentrations and slow HNO complex formation within the film limit this probe in its current state.
Acknowledgements We thank the National Science Foundation (CHE-061194) for support of research from our laboratory that was covered in this review.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
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Fluorescence-based nitroxyl sensing has been accomplished using conducting metallopolymers. Previous work produced NO-sensing CMPs containing copper(II)-binding oligopyridyl units incorporated into poly( p-phenyleneethynylene), poly( p-phenylenevinylene), poly(thiophene), and poly(fluorene) derivatives [32–34]. The CP complexes are quenched by the paramagnetic Cu(II), and the Cu(II)-CP complexes formed react with NO to reduce Cu(II) to Cu(I), thereby producing a fluorescent Cu(I)-CP species. These polymers exhibit competitive binding of Cu(II) between the intended metal-chelating bipyridyl units and the water-solubilizing side chains. When the bipyridyl units are replaced with bithiophene units the binding of Cu(II) to the CMP main chain is reduced [45]. The quenched Cu(II)-polymer becomes fluorescent upon exposure to excess NO in unbuffered solutions, but under simulated biological conditions results in a decrease in emission due to precipitation of the probe. When the same CMP is exposed to excess Angeli’s salt the fluorescence increases by 2.1-fold, making the polymer a selective HNO probe under simulated biological conditions.
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Conclusions and future directions
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The field of reactive nitrogen species sensing is an active and exciting area of research promoted by the increased interest into the role of RNS as signaling agents in biology. The NO-sensing domain has been improved by increasing the biocompatibility of probes such that tissue and animal experiments can now be considered. Peroxynitrite detection has been made possible by designing probes that react specifically with ONOO, instead of the standard ‘global-oxidation’ responsive sensors. A few metal-based systems that can discriminate HNO from NO have been described, allowing the field of nitroxyl detection to emerge. The major design feature that needs to be addressed is reversibility, which is required to get true spatiotemporal resolution. In addition, subcellular localization and ratiometric RNS detection, both of which have been achieved in cation sensing, would be valuable for determining signal localization and quantification, respectively. Current Opinion in Chemical Biology 2010, 14:43–49
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