Designing photoluminescent molecular probes for
singlet oxygen, hydroxyl radical, and iron
–oxygen
species
Youngmin You*aand Wonwoo Nam*b
Metabolism of molecular oxygen in the human body inevitably produces reactive oxygen species (ROS). Among the family of ROS, those exhibiting very high reactivities, including_OH, _OOH, ONOO, HOCl, HOBr, and1O
2, remain relatively underexplored. While emerging evidence suggests that these ROS are
critically involved in physiological function as well as the genesis and development of diseases, the scarcity of the knowledge about these species hampers full understanding of the pathophysiology caused by the oxidative metabolism. To unveil the molecular mechanisms of the ROS, photoluminescent probes capable of reporting identity, concentration, and trafficking of the individual class of the ROS are in great demand. In this Perspective, we focus on1O2and_OH and summarize the known principles for
designing photoluminescent molecular probes for these species. Special emphasis is placed on molecular design to selectively detect 1O2 and _OH. Disadvantages and limitations of using the
established probes for detection of biological1O2and_OH are discussed as well. Finally, we address the
importance of metal–oxygen species, such as iron–oxygen complexes of peroxo, superoxo, and oxo ligands, in oxidative processes and modulation of ROS. Despite the fact that the very high reactivity of the metal–oxygen species is recognized to be a key factor in many oxidative metabolic pathways, strategies for rational design of photoluminescent probes for detection of these species have yet to be established. As an initial step, we summarize the reactivities of the iron–oxygen species and propose potential approaches for creation offluorescent molecular probes in the future. The principles provided in this Perspective will be helpful for designing photoluminescent ROS probes capable of applications in the biological milieu.
Youngmin You received B.S. (Magna Cum Laude) and M.S. degrees in Chemical Engineering from Seoul National University, Korea. He then moved to the Department of Materials Science and Engineering at SNU and earned his Ph.D. in 2007. He became an Assistant Professor at Kyung Hee University in 2013. His research includes triplet-state photofunctionality of organic and organometallic molecules.
Wonwoo Nam received his B.S. (Honors) degree in Chemistry from California State University, Los Angeles and his Ph.D. degree in Inorganic Chemistry from UCLA under the direction of Professor Joan S. Valentine in 1990. Aer one year of post-doctoral experience at UCLA, he became an Assistant Professor at Hong Ik University in 1991. He
moved to Ewha Womans
University in 1994, where he is presently a Distinguished Professor of Ewha Womans University. His current research focuses on dioxygen activation, water oxida-tion, and sensors for metal ions in bioinorganic chemistry. aDepartment of Advanced Materials Engineering for Information and Electronics,
Kyung Hee University, Yongin, Gyeonggido 446-701, Korea. E-mail: odds2@khu. ac.kr
bDepartment of Chemistry and Nanoscience, Ewha Womans University, Seoul
120-750, Korea. E-mail: wwnam@ewha.ac.kr Cite this:Chem. Sci., 2014, 5, 4123
Received 3rd June 2014 Accepted 3rd July 2014 DOI: 10.1039/c4sc01637h www.rsc.org/chemicalscience
Science
PERSPECTIVE
Published on 04 July 2014. Downloaded by Ewha Womans University on 30/09/2016 07:23:59.
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Introduction
Reactive oxygen species (ROS) are metabolites of molecular oxygen produced by normal and abnormal redox functions in the human body. Assays for biological specimens and their model systems have identied production of ROS such as singlet oxygen (1O2), hypochlorous acid (HOCl) and hydroxyl
radical (_OH) and nitrogen-containing species such as nitric oxide (_NO). While these ROS are frequently recognized as toxins that cause aging1,2 and numerous pathological conditions,3–7 emerging studies have also related them with signaling func-tions.2,8–10This biological signicance of the ROS has sparked intensive research which has provided evidence that the indi-vidual species of ROS have unique properties and that ROS with high oxidizing power dominate many oxidative processes.11For example, Caldwell and co-workers suggested that production of peroxynitrite (ONOO) might be directly related to apoptosis of retinal vascular endothelial cells.12 In addition, oxidation of
DNA, proteins, and lipids is considered to be mainly caused by H2O2-derived_OH.13
The term‘highly reactive oxygen species’ rst appeared in the literature in 1977 to describe highly oxidizing endogenous molecules,14 and generally refers to 1O2,_OH, _OOH, ONOO,
HOCl, HOBr, and the reactive intermediates of metal–oxygen species. These ROS differ from other species, in that they are short-lived and their effects are extremely site-specic. These properties hamper direct monitoring of the ROS in vivo. Indeed, the nature of the ROS remains scarcely understood to date, and the research in this area has frequently been impeded by the lack of proper tools to detect the ROS. To facilitate such studies, reliable and efficient methods for the detection of the ROS are essential. Electron paramagnetic resonance (EPR) spectroscopy and assays of oxidation products have been employed for this purpose, but the currently existing methods pose signicant drawbacks for use in biological samples. Photoluminescence probes are ideal for circumventing the disadvantages encoun-tered by the current ROS detection methods, as they exhibit spatiotemporal resolutions and sensitivity suitable for real time monitoring of living specimens. In addition, judicious design of probe platforms enables ratiometric detection for quantitation of ROS. Moreover, the available probe library can be greatly expanded by taking advantage of the accumulated knowledge about synthetic tailoring of photoluminescence properties. Thus, it is very appealing to create photoluminescent probes that can provide information about identity, concentration, and trafficking of the individual species of the ROS.
Development of photoluminescent ROS probes has been retarded by the difficulties in devising sensitive and selective sensor platforms. These difficulties arise from the transient nature of the ROS, due to their high reactivities and the rapid and complex interconversion reactions that can occur within the family of ROS. Although the chemical mechanisms for production of the ROS and their interconversions are not fully understood, it is generally agreed that O2_and H2O2 serve as
sources of the majority of the ROS. While O2_ is primarily
produced due to electron leakage during the mitochondrial
electron transport chain, other mechanisms, including enzy-matic processes, auto-oxidation of biological molecules, and disruption of heme Fe(II)–O2adducts can yield O2_in vivo.13,15
Photoinduced one-electron reduction of O2 by endogenous
photosensitizers (i.e., Type I reaction) is also responsible for O2_
generation. Once generated, O2_ quickly undergoes
dis-mutation into H2O2 by superoxide dismutases (SOD).
Mean-while, despite lower probabilities, O2_ can react with H2O2,
HOCl,_OH, and _NO to yield _OH,161O
2,17_OOH,18and ONOO,19
respectively (see Scheme 1 for details). In addition, protonation equilibrates O2_with HOO_ with a pKavalue of 4.8.15Relatively
long-lived H2O2 is a better precursor of the ROS with high
oxidation power. H2O2 is reductively cleaved to_OH by Fe(II) (Fenton reaction) or Cu(I) ions. In the presence of Cland Br, H2O2 is converted to HOCl4,20 and HOBr,21 respectively. The
resulting HOCl can serve as a source for_OH by the catalytic action of Fe(II) ions.13It has also been suggested that O2_is
regenerated from H2O2by Fe(III) ions22or upon the reaction with
_OH.23Generation of1O
2from H2O2and ClO(or BrO) has been
reported although the biological relevance of the reactions has not been veried.13Ferric complexes react with H
2O2to yield an
iron(III)–hydroperoxo species, which is subsequently converted to an iron(IV)–oxo or an iron(III)–peroxo species with high oxidation capabilities.24Alternatively, the high-valent iron(IV)–
oxo and iron(III)-peroxo species can be generated by the inter-actions of the ferrous centers of heme and non-heme proteins with HOCl or HOBr.24
As demonstrated by the conversions shown in Scheme 1, it is extremely difficult to recognize an absolute dichotomy between sources and products among the ROS. This poses major obstacles in elucidating which individual species in the family of the ROS actually serves as a true oxidant in the reactions of interest. Furthermore, due to the high oxidizing capability and limited diffusivity in the biological milieu, the diffusion lengths of some ROS are signicantly short (Table 1). For instance, the diffusion length of_OH is shorter than 20 mm, being ca. 75 times smaller than that of H2O2.25 This spatially and temporally
transient nature presents a daunting challenge to design sensor platforms for detection of the ROS.
In this perspective, we focus on the molecular design strat-egies for the construction of photoluminescent probes to detect
1O
2,_OH, and the highly oxidizing iron–oxygen species. These
species are excellent examples showing the distinct nature of the ROS because, besides the high reactivity, their reactions are highly selective (1O2 and iron–oxygen species) or exceedingly
uncontrolled (_OH). We give an overview of the scope of reac-tions mediated by1O2,_OH and iron–oxygen species, and then
summarize the known principles of photoluminescent molec-ular probes of these ROS. A particmolec-ular focus is placed on signaling and kinetic selectivity toward detection of the indi-vidual species.
Singlet oxygen probes
Biological1O2 (1Dg) is produced through triplet
photosensiti-zation under aerobic conditions.32 1O
2 is also generated by
several biochemical processes without photoirradiation; H2O2,
O2_and HOCl are enzymatically converted to1O2(Scheme 1),
where myeloperoxidase, lactoperoxidase, chloroperoxidase, lipoxygenase, and horseradish peroxidase catalyze such conversion.33–39Although1O2is higher in energy than3O2by 94
kJ mol1,28 the capability of 1O2 for one-electron oxidation
remains low. The reason for1O2being a highly reactive oxygen
species should be ascribed to the relief from the spin-restriction for reactions with ground-state (i.e., singlet) substrates. In fact,
1O
2 is a strong dienophile toward the [2 + 4] cycloaddition
reactions with a variety of biomolecules having cisoid-diene structures. Nucleic acids are the major targets of1O2; it has been
demonstrated that the cycloaddition reaction between1O 2and
guanine yields an unstable endoperoxide intermediate that subsequently undergoes further reactions to produce several oxidation products, including carbamic acid (Scheme 2).40In
addition to guanine, amino acids, such as tryptophan, tyrosine, and histidine, are also excellent substrates for1O2.41Studies for
the model systems of the dienyl amino acids established that the oxidation reactions proceed via endoperoxide or
hydroperoxide intermediates, with the rate constants as large as 106–107M1s1.41
1O
2can be directly detected by monitoring its
phosphores-cence emission at 1268 nm. However, this method is of narrow practical utility, because the photoluminescence quantum yield of the 1268 nm emission is lower than 106.28In addition, short half-lives (s1/2< 10ms) of1O2prohibit phosphorescence
detec-tion with signal-to-noise ratios appropriate for bioimaging.26
Thus, biological studies on1O
2rely on the use of probe
mole-cules. 1O2 probes suitable for biological applications should
satisfy several requirements: the response should be fast, sensitive, and selective to1O2. In addition, the probes should
not alter the local 1O2 levels under photoexcitation. These
criteria can be met by mimicking the biochemical reactions of
1O
2summarized in Scheme 2. A variety of dienyl compounds,
such as anthracene, imidazole, furan, and isobenzofuran, have thus been incorporated into chromophoric platforms to create photoluminescent 1O2 probes. These dienes undergo [2 + 4]
cycloaddition reactions with1O2, producing endoperoxides with
alteredp-conjugation lengths or electrochemical potentials. The development of uorescent, biologically useful 1O2
probes has been pioneered by Nagano and co-workers. The Nagano group displaced the bottom phenyl ring ofuorescein with 9,10-disubstituted anthracene to prepare the1O2 probes
(1 and 2 in Scheme 3).42,43 The anthracene moieties undergo
cycloaddition with1O2 at rate constants of 106–107M1s1.44
The probes exhibiteduorescence turn-on responses, because the formation of endoperoxide effectively suppressed the non-radiative photoinduced electron transfer occurring between Scheme 1 Production and consumption of the reactive oxygen species.
Table 1 Lifetimes, diffusion lengths and in vivo concentrations of1O2
and_OH
Lifetime Diffusion length In vivo concentrations
1O
2 1–10 ms (ref. 6 and 27) <1 mm (ref. 29) N.A.
0.04ms (ref. 28) 20 nm (ref. 28)
_OH 1 ns (ref. 13 and 30) <20 mm (ref. 25) 0.05–400 mM (ref. 31)
uorescein and the anthracene unit. The sensing mechanism involving photoinduced electron transfer was supported by laserash photolysis, which identied formation of the radical anion species of anthracene. Incorporation of anthracene into the 20-position ofuorescein yielded another uorescence1O2
probe (3).45 Although the probe was cell-impermeable, it
exhibited hugeuorescence turn-on responses in the biological milieu. However, several drawbacks in using the probe were observed: theuorescence response was slow and sensitive to pH changes.42Most of all, the chlorinateduorescein produced
1O
2, causing overestimation of 1O2 levels in the regions of
interest.46 Although tetrauorination of xanthene could Scheme 2 Biological oxidation reactions of1O2.
Scheme 3 Photoluminescent probes for detection of1O2(red structures indicate 1
O2-reactive moieties).
attenuate photogeneration of1O2,47this drawback still remains
to be resolved.
The ability of acenes to trap1O2prompted creation of several
other classes of photoluminescent1O
2probes. Probes
exhibit-ing phosphorescence (4) and long-lived photoluminescence (5) have been constructed based on Re (ref. 48) and Eu (ref. 49) complexes, respectively. In an alternative approach, tetracene exhibiting red uorescence emissions was incorporated into lms of strongly blue-uorescent poly(uorene) (7).50 The
polymerlms exhibited sensitized red emission from the doped tetracene molecules upon the selective photoexcitation of pol-y(uorene), whereas1O
2reactions abolished such sensitization.
To maximize energy transfer, poly(phenylene-co-uorene) teth-ering 5,12-diarylated tetracene (6) and 2,5-diphenylfurane units (9) were prepared.51,52 In addition to the acene derivatives,
dienyl heterocycles can also serve as effective1O
2 traps. Tang
and co-workers attached histidine onto a NIR-uorescent cyanine dye (8).53Fluorescence quenching most likely due to
photoinduced electron transfer from histidine to cyanine was observed, but the cycloaddition reaction at imidazole restored theuorescence emission of cyanine. The uorescence turn-on utility was further demonstrated for visualization of endoge-nously produced1O2 in RAW264.7 cells under stimulation by
phorbol 12-myristate 13-acetate (PMA).
Among the diene compounds, isobenzofuran is ideal for construction of 1O2 probes, as it exhibits fast response (rate
constant¼ 9.6 108M1s1) and unitary photoluminescence quantum yields.54Most of all, intersystem crossing is so slug-gish in isobenzofuran that photosensitization of 1O2 can be
suppressed.55 Actually, 1,3-diphenylisobenzofuran (DPBF) has been widely employed as a1O2trap.56Decreases in the UV-vis
absorbance (labs ¼ 415 nm, log 3 ¼ 4.26) and uorescence
intensity (lem¼ 455 nm) of DPBF are unambiguous indications
for the presence of 1O
2. To create uorescence ratiometric
probes, we modied the structure of DPBF by replacing one phenyl ring with variousuorophores (10–13).57The modied
isobenzofuran probes reacted with 1O
2 to produce unstable
endoperoxides, where the weak O–O bond was readily broken to generate 1,2-diketone compounds exhibiting hypsochromically shied uorescence emission. The ratiometric uorescence changes were thus observed, which promises quantication of biological1O2.
Chemoluminescent probes that do not require photoexcita-tion have been developed for detecphotoexcita-tion of1O2. The principle for
chemoluminescent detection involves the [2 + 2] cycloaddition reaction between1O2and alkene to produce 1,2-dioxetane. The
1,2-dioxetane is cleaved by certain chemical stimuli, yielding an excited-state species. McNeill and co-workers prepared a steri-cally congested alkene compound consisting of adamantane, a methoxy group, and a silyl-protected 3-phenol (14 in Scheme 4).58Reaction with1O
2furnished a thermally stable
1,2-dioxe-tane. Subsequent addition ofuoride anion removed the silyl group, facilitating breakage of the 1,2-dioxetane into a singlet excited state of adamantone and methyl 3-hydroxybenzoate (Scheme 4). Strong chemoluminescence was generated from the former. trans-Diethoxyethene also served as a chemo-luminescent probe for 1O2 (15).59 In this case, the resulting
diethoxy-1,2-dioxetane was cleaved by 9,10-dibromoanthracene-2-sulfonic acid. Similarly, electron-rich tetrathiafulvalene was employed for chemoluminescent detection of1O2. The group of
Ma, Zhang and Zhu demonstrated the capability of a molecular dyad consisting of tetrathiafulvalene and anthracene for 1O
2
sensing (16),60although the proposed mechanism for the1O 2
responses requires further resolution.
The sensing properties of the1O2 probes are compiled in
Table 2. The examples summarized here demonstrate that cycloaddition reactions can serve as useful principles for construction of photoluminescent 1O2 probes. However, the
previous1O2probes lacked the full reversibility because
endo-peroxides are unstable, and cycloreversion is kinetically forbidden. Thus, theuorescence signals from the probes could not provide any quantitative information about the1O2levels.
Hydroxyl radical probes
Among the various classes of the ROS,_OH is considered as the most damaging species.3,13_OH can oxidize almost all
biomole-cules, including carbohydrates, proteins, and nucleic acids. Oxidative damage of these biomolecules can lead to disorder in cellular regulation, which provides potent mechanisms for the genesis and development of fatal diseases, such as cancer, cardiovascular disorder, atherosclerosis, and neurodegenera-tive diseases.3,61The high reactivity of_OH can be ascribed to a very large reduction potential (Eored(_OH/HO)¼ 1.77 V)62as well
as to the radical character. Actually, the oxidizing power of_OH is proposed to be greater than that of ONOO.63Due to the high reactivity, the lifetime of _OH remains less than a few nano-seconds in aqueous solutions,9,30and physiological concentra-tions of_OH are highly site-specic. Furthermore, as shown in Scheme 1, the complex interconversion reactions between_OH and other ROS lead to an ambiguity whether the observed phenomena are due to_OH or different species derived from_OH. Accordingly, the nature of_OH in biological reactions remains poorly understood to date.
Conventionally, EPR spectroscopy has been employed for detection of_OH. A variety of spin-trapping agents, including Scheme 4 Chemoluminescent probes for detection of1O2.
5,5-dimethyl-1-pyrrolidine-N-oxide (DMPO), hydroxyl-2,2,6,6-tetramethylpiperidine-N-oxide (TEMPO), a-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN), and a-phenyl-N-tert-butylnitrone (PBN), are used to improve the signal-to-noise ratios of the EPR signals.64Despite the capability for in vivo imaging, the utility of the EPR detection is frequently limited by low spatiotemporal resolutions and expensive instrumentation. Thus, the current methods for identication of _OH rely on molecular dosimeters, including salicylate,65phenylalanine,66nitrophenol,67and tere-phthalate.68These molecules undergo aromatic hydroxylation with_OH, and the hydroxylated products can be analyzed by HPLC and spectroscopic techniques. Among the_OH dosime-ters, use of terephthalate (TA) is preferred, because hydroxyl-ation of TA is ultrafast (rate constant¼ 3.3 109M1s1)69and
yields a single, uorescent product, 2-hydroxyterephthalate (HTA;lem¼ 425 nm). Furthermore, contrary to salicylate and
phenylalanine, TA and HTA are not metabolized in vivo. Several groups have demonstrated the utility of TA for uorescent detection of_OH produced during microdialysis68 and under photoirradiation of hair melamine.70
To facilitate_OH detection, probes with improved photo-luminescence properties have been created based on aromatic hydroxylation. Coumarin-3-carboxylic acid (CCA;17 in Scheme 5) is auorescent _OH probe that is compatible with conven-tionaluorescence microscopy. Aromatic hydroxylation occurs exclusively at the 7 position of CCA, yielding strong uores-cence.71Pierre and co-workers developed a novel system
con-sisting of a Tb complex and trimesate, a _OH-reactive pre-antenna (18 in Scheme 5).72 The_OH detection mechanism
involved_OH-mediated hydroxylation of trimesate, followed by coordination of the hydroxylated antenna to the Tb center. The complexation evoked an increase in photoluminescence from the 5D4 state of Tb3+, because the hydroxylated trimesate
effectively transferred its excited-state energy to the Tb center (Table 3).
The strong propensity of_OH for hydrogen atom abstraction has been utilized to prepareuorescent probes. The reduced leuco forms of uorophores, such as 2,7-dichlorodihydro-uorescein (DCFH) and dihydrorhodamine, can be readily oxidized by _OH (19 and 20 in Scheme 6).73,74 These pro-uorescent probes become pro-uorescent upon interactions with _OH, as the p-conjugation networks of xanthene are recovered through hydrogen atom abstraction, followed by one-electron oxidation. The approach based on reduced leuco forms of dyes is reminiscent of a uorescent O2_ probe, dihydroethidium
(DHA).75 Since many uorescent dyes can be reduced using
typical hydride sources, a wide range of prouorescent _OH probes would be available. Murthy and co-workers synthesized hydrocyanine derivatives (21 in Scheme 6) by reducing the Table 2 Sensing properties of the1O
2probes listed in Schemes 3 and 4
Signal type labsa(nm) lema(nm) PLQYa,b kc(M1s1) Cell-permeability
1 Turn-on 492 515 0.81 2.0 107 — 2 X¼ H Turn-on 494 515 0.53 — — X¼ Cl Turn-on 506 527 0.66 — — X¼ F Turn-on 494 515 0.70 — — 3 Turn-on 507 536 0.43 3.8 108 — 4 Turn-on 390 555 0.00071 — — 5 Turn-on 400 614 0.14 4.4 1010 Yes 6 m¼ 0 Ratiometric 300–400 510/ 417 0.58d — — m¼ 1 Ratiometric 300–400 422/ 417 0.44d — — 7 Ratiometric — 510/ 420 — — — 8 Turn-on 754e 794 — — Yes 9 X¼ H Turn-off 335 417 0.58d — — X¼ CF3 Turn-off 346 415 0.58d — — 10 Turn-off 298 — — 9.6 108 — 11 Ratiometric 324 476/ 370 0.0078 2.1 109 f — 12 Ratiometric 358 512/ 398 0.061 1.4 1010 f Yes 13 Ratiometric 382 505/ 435 0.41 2.4 1010 f — 14 Turn-on — — Chemoluminescence 2.1 103s1 g — 15 Turn-on — — Chemoluminescence — — 16 Turn-on — 420 Chemoluminescence — —
aValues aer the reaction with1O
2.bPhotoluminescence quantum yield.cRate constant for the reaction with1O2.dValues before the reaction with 1O
2.eExcitation wavelength.fEstimated relatively from the known value for10.gAt 100mM probe and an excess amount of1O2.
Scheme 5 Photoluminescent detection of_OH based on aromatic hydroxylation.
iminium moiety of cyanine with NaBH4.76Huge uorescence
turn-on responses in the NIR region were observed upon the reaction with_OH. The Murthy group further demonstrated the utility of the probe by visualizing endogenously produced_OH in rat aortic smooth muscle cells and tissues upon stimulation by angiotensin (Ang) II and lipopolysaccharide (LPS), respectively. In addition, the strong NIR uorescence allowed for in vivo imaging of _OH generated in a LPS model of acute inamma-tion. To create a ratiometricuorescence probe for_OH, Lin and co-workers adopted a similar strategy.77The probe comprised
coumarin and half the structure of cyanine (22 in Scheme 6).
The reduced leuco form of the probe showed uorescence emission of coumarin atlem¼ 485 nm, whereas the addition of
_OH provoked bathochromically shied cyanine uorescence at lem¼ 651 nm.
Great caution is, however, required in the use of the pro-uorescent probes because they are prone to auto-oxidation and photobleaching. Further, the oxidation reactions show poor selectivity for_OH over other ROS. For example, hydrocyanine can also be oxidized to cyanine by O2_.76In addition, some
cyanine dyes with low oxidation potentials are susceptible to further oxidation to produce nonuorescent oxindole.78Nagano
and co-workers have exploited this oxidative fragmentation, and employed the behavior to create auorescence ratiometric ROS probe that consisted of two cyanine dyes with differing reactivity toward ROS.78
The radical character of_OH suggests use of persistent radicals for_OH recognition. It has been demonstrated that conjugation of nitroxide (R2N–O_), such as TEMPO, to
uo-rophores abolishes uorescence emission,79 whereas dia-magnetization by reactions between nitroxide and any reactive radical species can restore theuorescence inten-sity. This principle provides a promising strategy to construct uorescence turn-on probes for _OH. Since a wide range of spin-trapping agents has been established for the EPR tech-niques (vide supra), the nitroxide–uorophore dyad approach can benet from the established library. Accordingly, several uorophores, including anthracene (23),80 BODIPY (24),81
uorescamine (25),82 and rhodamine (26),83 have been
conjugated to nitroxide to prepare uorescent _OH probes (Scheme 7). As expected, these probes could report the presence of chemically and enzymatically produced_OH by uorescence turn-on signaling, and some of them were successful in visualizing intracellular _OH in macrophage cells81and oxidative stress models.82
Table 3 Sensing properties of the_OH probes listed in Schemes 5–9
Signal type labsa(nm) lema(nm) PLQYa,b kc(M1s1) Cell-permeability
17 Turn-on 380–400d 450 0.50 5 109 — 18 Turn-on 333d 545 — 5 109 — 19 Turn-on 504 515 0.85 2.8 109 Yes 20 Turn-on 506 529 0.90 — Yes 21 n¼ 1 Turn-on 535d 560 — — Yes n¼ 2 Turn-on 635d 660 — — — n¼ 3 Turn-on 735d 760 — — Yes 22 Ratiometric 460d 485/ 651 — 5.14 104s1 e — 23 Turn-on — 430 — — — 24 Turn-on 560 601 0.20 — Yes 25 Turn-on — 490 — — — 26 Turn-on — 590 — — Yes 27 R¼ O Turn-on f f f — Yes R¼ NH Turn-on f f f — Yes 28 R¼ O Turn-on g g g — Yes R¼ NH Turn-on g g g — — 29 Turn-on 320d 488, 541, 581, 618, 642 0.078 — Yes 30 Ratiometric 496 576/ 518 — — —
aValues aer the reaction with_OH.bPhotoluminescence quantum yield.cRate constant for the reaction with_OH.dExcitation wavelength.eAt 20
mM concentration of the probe.fSpectral properties are those ofuorescein.gSpectral properties are those of rhodamine.
Scheme 6 Profluorescent probes of _OH based on reduced leuco forms of dyes.
However, the probes based on nitroxide required DMSO for their operation, because radical coupling proceeded only with _CH3 that was derived from_OH and DMSO (Scheme 7).84The
rate constant for the radical coupling reaction is as large as 7.8 108M1s1.82Blough and co-workers investigated the
reaction of auorescamine-derived probe with _OH produced from the Haber–Weiss reaction and a diaziquinone-based anticancer drug.82The_OH detection required 750 mM DMSO (phosphate buffer at pH 7.5), indicating that the response was actually due to_CH3. The Blough group further identied an
O-methylated hydroxyl amine product. Rosen and co-workers directly compared the responses of theuorescamine-derived probe toward O2_(produced from xanthene oxidation),_OH
(FeSO4 + xanthene + xanthene oxidase), and_CH3 (FeSO4 +
xanthene + xanthene oxidase + 0.14 M DMSO).85 In an
air-equilibrated Hank's balanced salt solution containing 0.1 mM diphenylenetriaminepentaacetic acid, the probe showed uo-rescence turn-on responses to_CH3. This result is consistent
with the mechanism that the nitroxide cannot react directly with_OH. Although DMSO is considered relatively nontoxic and its reaction with_OH is ultrafast (rate constant ¼ 7.0 109
M1 s1),84 the requirement of high DMSO concentrations does nott the conditions for _OH bioimaging applications. Another disadvantage of employing nitroxide is that it can be metabolized to hydroxylamine by GSH and NADPH.85 Such reduction can also be proceeded by ascorbic acid.85 Accord-ingly, the uorescence probes based on nitroxide can over-estimate the_OH levels.82Furthermore, signaling mediated by _CH3 is easily affected by side reactions with O2 due to
production of the reactive methylperoxo radical (CH3OO_).
Indeed, Gu and co-workers employed CH3OO_ in the Hantzsch
reaction for uorescent detection of _OH.86 The reaction
involved disproportionation of CH3OO_ into HCHO, followed
by the Hantzsch reaction with 1,3-cyclohexanedione to produce auorescent product. Since the Hantzsch cyclization required high temperatures for completion, biological utility of this method would be low.
The strong propensity of_OH for ipso-substitution of elec-tron-rich aromatics can provide a valuable principle for construction of uorescent _OH probes. Aryloxy groups con-taining p-hydroxyl and p-amino substituents are susceptible to attack by_OH (Scheme 8). Hydroquinone and 4-aminophenol were tethered through aryloxyether linkages to uorophores, such asuorescein (27)87and rhodamine (28).88These aryloxy groups facilitated photoinduced electron transfer upon photo-excitation of the vicinaluorophores to quench uorescence emission. Removal of the aryloxy moieties by_OH restored the inherentuorescence intensity of the uorophores. One benet of using the probes based on this mechanism is the improved stability against auto-oxidation. The principle was further utilized to create photoluminescent probes based on lanthanide complexes (29 in Scheme 8).89It should, however, be noted that
the responses of the probes by ipso-substitution were not exclusive to_OH; HOCl and1O
2were reported to produce
iden-tical signaling although the reaction mechanism requires further resolution.90
DNAs are vulnerable to oxidative damage mediated by_OH. It has been well documented that deoxyribose in DNAs undergoes the hydrogen atom abstraction reaction by_OH to yield abasic sites.91Although the cleavage occurs in a sequence-independent manner, the reaction by_OH proceeds faster than the cleavage by nucleases. By taking advantage of the oxidative cleavage of DNAs, several approaches touorescent detection of _OH have been developed. Imato and co-workers synthesized a deoxy-thymidine dimer (TT) having uorescein and rhodamine at both its termini (Scheme 9).92
Photoexcitation (lex ¼ 496 nm) of the uorescein moiety
promoted sensitized uorescence emission from rhodamine (lem¼ 576 nm) due to efficient uorescence resonance energy
transfer._OH produced under the Fenton conditions (i.e., FeSO4
+ H2O2) broke the TT linker, putting the twouorophores apart.
Accordingly, photoexcitation at lex ¼ 496 nm produced the
inherentuorescence emission from the uorescein (lem¼ 518
nm). This idea was utilized by Tang and co-workers to prepare Scheme 7 _OH probes containing spin traps.
an Au nanoparticle (Au NP)-baseduorescent probe for _OH.93 The Tang group linked Au NPs anduorescein through single-stranded DNA (ssDNA). The Au NP effectively quenched the uorescence emission of uorescein. Chemically generated _OH cleaved the ssDNA, resulting inuorescence turn-on responses. This probe was permeable to macrophages and liver cancer cells, and allowed foruorescent visualization of endogenously produced_OH. The group of Zhang and Tan prepared a molec-ular beacon where auorescence quencher and a uorophore were attached at the two ends of a single-stranded oligonucle-otide with a hairpin structure.94This structure placed the two photofunctional components in close proximity, resulting in improved quenching efficiencies. Fluorescence turn-on responses were observed, as the molecular beacon was dis-rupted by treatments with enzymatically produced_OH.
Probes for metal
–oxygen species
Metalloenzymes activate dioxygen to carry out a variety of bio-logical reactions, including biotransformation of naturally occurring molecules, oxidative metabolism of xenobiotics, and oxidative phosphorylation. The dioxygen activation at the active sites of the metalloenzymes occurs through several steps, involving the generation of metal–superoxo and metal–peroxo species by binding of O2 and the O–O bond cleavage of the
metal–dioxygen species to form high-valent metal–oxo inter-mediates.95–101 Alternatively, ROS are harvested to form the metal–oxygen species (Scheme 1). For instance, the recent mechanistic studies based on mass analyses provided compel-ling evidence that the Fenton reaction may involve Fe(IV)–oxo species as the key oxidizing intermediate.102 It has been demonstrated in biomimetic studies that such metal–oxygen complexes are key intermediates in the catalytic oxidation of biological substrates.24,98,103–111 These metal–oxygen species release ROS during the course of the catalytic cycle of oxidation reactions. For example, the iron–superoxo and iron–peroxo species release O2_and H2O2in cytochrome P450.112It should
also be noted that intracellular levels of ROS and metal–oxygen species are mutually balanced, as seen from down regulation of CYP1A1 expression by ROS.112These complex actions of metal–
oxygen species with ROS, together with the high catalytic activity in oxidation reactions, frequently hamper the capture
and identication of true oxidants. It was observed that DCFH (19 in Scheme 6) produced uorescence responses upon direct interaction with hemin and cobalt–protoporphyrin, a heme oxygenase-1 inducer.73 The extent of uorescence turn-on of DCFH was proportional to the intracellular level of hemoglobin and was unaffected by the addition of catalase and SOD. These results indicate that the observeduorescence response did not originate from oxidation by H2O2, O2_, and other ROS derived
from these species. Therefore, it is necessary to consider the function of metal–oxygen species for oxidation and site-specic ROS regulation in the study of biological ROS.
Surprisingly, uorescence techniques have been rarely utilized in the chemical studies of oxophilic metalloproteins and their model compounds. The few examples include use of intrinsic tryptophanuorescence to monitor global conforma-tion changes of human H-chain ferritin113and a non-heme Fe(II) enzyme, 2,4-dichlorophenoxyacetic acid/a-ketoglutarate dioxy-genase, TdfA.114Tryptophanuorescence was also employed to probe the intermediate states of cytochrome oxidase in the generation of transmembrane ion gradients,115 and to
deter-mine the redox state of the non-heme iron center of human 5-lipoxygenase.116Meanwhile, the electron transfer kinetics in a
model system consisting of red-uorescent AlexaFluor 657 and cytochrome b5 (Cytb5) were investigated, with a focus on
photoinduced reduction of Fe(III) to Fe(II) of the heme in Cytb5.117Fundamental aspects of intramolecular energy transfer
from naphthalene to oxo-bridged dinuclear Fe(III) complexes have also been characterized.118 Despite these studies, the development ofuorescent probes capable of detection of bio-logical metal–oxygen species remains unexplored. This scarcity may originate from the high reactivity of the metal–oxygen species in nature and the difficulty in directing the probes in the vicinity of the metal center.
To address this challenge, judicious probe design that enables fast and selective detection of the metal–oxygen species is essential. One viable approach is to take advantage of the biomimetic oxidation reactions of the model compounds of metal–oxygen species. A wide variety of model compounds mimicking the oxidative metabolism of redox-cycling metal-loenzymes have been established to date.103–111,119–124In
partic-ular, studies on the synthetic models of the heme and non-heme iron enzymes provided many aspects of the structures, Scheme 8 Fluorescent probes based onipso-substitution of _OH.
chemical properties, and reactivities of oxidizing intermediates in the catalytic cycles.24,103,119,120 It has been established that
interaction of the iron-containing model complexes with ROS, such as H2O2, generates the Fe(III)–hydroperoxo (Fe(III)–OOH) species that subsequently undergoes heterolysis or homolysis to yield a Fe(IV)–oxo species.125 Deprotonation of the Fe(III)–OOH produces Fe(III)–peroxo. Alternatively, direct O2binding at the
iron center provides Fe(IV)–oxo compounds through an O2
-bridged dinuclear Fe(III) intermediate. In addition, Fe(IV)–oxo species are generated from Fe(II) complexes upon interactions with ROS, including O3, NaOCl and NaOBr (Scheme 1). It should
be noted that these iron–oxygen species, such as Fe(III )–super-oxo, Fe(III)–peroxo, Fe(III)–OOH, and Fe(IV)–oxo complexes, display different scopes toward oxidation reactions. For instance, Fe(IV)–oxo species can catalyze N-dealkylation, aromatic hydroxylation, phosphine oxidation, epoxidation, sulfoxidation, aliphatic hydroxylation, and alcohol oxidation, while the Fe(III)–peroxo species undergoes nucleophilic reac-tions, such as deformylation of aldehydes.24,125A summary of
the iron–oxygen species in oxidation reactions is shown in Scheme 10.
The knowledge about orthogonality in the reactions of the individual species of metal–oxygen compounds may allow for the design of highly selective uorescent probes that are appropriate for biological applications. For instance, the formyl group can be incorporated into the p-framework of uo-rophores for selective identication of Fe(III)–peroxo species.
Since variousuorophores that are compatible with biological systems have been established to date, a diverse range of uo-rescent probes can be designed and employed for detection of biological metal–oxygen species. Combined with appropriate targeting methods, the probes will unveil the mechanisms and effects of oxidative pathophysiology.
Summary and outlook
Emerging evidence strongly suggests that ROS, such as1O2and
_OH, play important roles in oxidative pathophysiology in the human body. However, understanding of the biochemical mechanisms of these ROS is at the beginning stage due to lack of proper tools for identication, quantitation, and spatiotem-poral monitoring of these species. Thus, the development of photoluminescent molecular probes for the ROS is of substan-tial importance. In this perspective, we gave an overview of the molecular principles for the construction ofuorescent probes for detection of1O2and_OH. The dienophilic character of1O2
has been combined with various photophysical functions to prepare biologically usefuluorescent1O
2probes. Although the
previous approaches relied mainly on the irreversible Diels-Alder type reactions, exploitation of the reactivities of1O
2would
provide novel insights into the development of reversible1O2
probes. The uncontrolled, highly oxidizing properties of_OH set a more daunting challenge to the creation ofuorescence _OH probes. Several principles have been utilized to synthesize_OH probes, including aromatic hydroxylation, hydrogen atom abstraction and subsequent one-electron oxidation, radical coupling, and ipso-substitution. It should, however, be noted that utility of these principles is limited, as other ROS and metal–oxygen species can sometimes produce identical signaling. In addition, kinetic selectivity of the probes toward _OH has been poorly explored. These limitations emphasize the requirement of future studies to devise molecular strategies for selective and reliable detection of_OH. Although the human pathophysiology by the metal–oxygen complexes (e.g., metal– superoxo, metal–peroxo, metal–hydroperoxo, and high-valent metal–oxo species) remains to be established rmly, the species are critically involved in the oxidation of biomolecules and the modulation of the ROS. Probes capable of detecting the metal– oxygen species over ROS are thus urgently demanded. The accumulated knowledge about the scopes and reactivities of the Scheme 9 Ratiometricfluorescence probe based on a _OH-reactive DNA linker.
Scheme 10 Scopes of the oxidation reactions mediated by iron– oxygen species.
metal–oxygen species may lead to potential structures of uo-rescent probes. In particular, the unique reactivities of the biomimetic iron–oxygen species (e.g., aldehyde deformylation) can provide novel probe platforms that are selective over_OH and other ROS with electrophilic character. Finally, since ROS are short-lived and their effects are site-specic, the strategy for designing of the ROS probes should involve proper methods for delivery of probes at the region of interests. Other conditions, such as brightness, dynamic ranges, and minimized alteration of local environments under photoexcitation, should also be met. We hope that the molecular principles summarized in the perspective will provide useful insights into the future devel-opment of ROS probes for bioimaging and therapeutic applications.
Acknowledgements
Authors acknowledgenancial support from the NRF of Korea through CRI 2012R1A3A2048842 to W.N.) and GRL (NRF-2010-00353 to W.N.) programs and a grant from Samsung Research Funding Center for Future Technology (SRFC-MA1301-01 to Y.Y.). Helpful discussions and suggestions by Professor Joan S. Valentine at Ewha Womans University are greatly appreciated.
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