Understanding the Selectivity of a Multichannel Fluorescent Probe for Peroxynitrite over Hypochlorite

Article abstract of DOI:10.1021/acs.bioconjchem.5b00396

Peroxynitrite is a prominent biological reactive nitrogen species from radical combination of nitric oxide and superoxide and fundamentally involved in broad spectrum physiological and pathological processes. Though redox-inert itself, peroxynitrite anion (OONO^-) attacks various biological electrophiles to generate an array of potent 2-e^- or 1-e^- oxidants, which result in cell injuries. Development of fluorescent probes for peroxynitrite, free from interference from hypochlorite, has been an active endeavor of the chemical community. We previously reported a peroxynitrite probe (PN600), which could differentiate hypochlorite from peroxynitrite through a multichannel signaling mechanism. Herein, this intriguing selectivity was accounted for through a structure-reactivity relationship study. Also, this work, together with rich literature contributions, has allowed a qualitative guideline in the use of electron-rich aromatic moieties to design probes against peroxynitrite and/or hypochlorite. The viability of this guideline was further testified by development of another list of peroxynitrite selective probes.

Full text of DOI:10.1021/acs.bioconjchem.5b00396

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Bioconjugate Chemistry
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Understanding the Selectivity of a Multi-Channel Fluorescent Probe for
Peroxynitrite Over Hypochlorite
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Quanjuan Zhang,† Na Zhang,§ Yi-Tao Long,§ Xuhong Qian†,‡ and Youjun Yang†,‡,*
† State Key Laboratory of Bioreactor Engineering, ‡ Shanghai Key Laboratory of Chemical Biology, School of
Pharmacy, § Department of Chemistry, East China University of Science and Technology, Meilong Road 130,
Shanghai 200237, China
youjunyang@ecust.edu.cn
ABSTRACT: Peroxynitrite is a prominent biological reactive nitrogen species from radical combination of nitric
oxide and superoxide and fundamentally involved in a broad spectrum physiological and pathological processes.
Though redoxꢀinert itself, peroxynitrite anion (OONOꢀ) attacks various biological electrophiles to generate an
array of potent 2ꢀeꢀ or 1ꢀeꢀ oxidants, which result in cell injuries. Development of fluorescent probes for
peroxynitrite, free from interferences from hypochlorite, has been an active endeavor of the chemical community.
We previously reported a peroxynitrite probe (PN600), which could differentiate hypochlorite from peroxynitrite
through a multiꢀchannel signaling mechanism. Herein, this intriguing selectivity was accounted for through a
structureꢀreactivity relationship study. Also, this work, together with rich literature contributions, has allowed a
qualitative guideline in the use of electronꢀrich aromatic moieties to design probes against peroxynitrite and/or
hypochlorite. The viability of this guideline was further testified by development of another list of peroxynitrite
selective probes.
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INTRODUCTION
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Peroxynitrite (OONOꢀ) biology dates back to 1986 when superoxide (O2 ) was shown to modulate the
halfꢀlife of the endothelial derived relaxing factor (EDRF), which was identified to be nitrogen monoxide (or
nitric oxide, NO) next year.1ꢀ6 Formation of OONOꢀ from nearꢀdiffusion controlled radical combination of NO
•ꢀ
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•ꢀ
and O2 was regarded as merely a cellꢀdetoxification mechanism of NO at the time, until Beckman suggested in
1990 that potent oxidants far more toxic than NO and “similar to hydroxyl (HO•) in reactivity” were effectively
derived from OONOꢀ.7ꢀ10 Much of the apparent oxidative cytotoxicity of NO, i.e. antibacterial and antimicrobial
activities, dysfunction of mitochondrial respiratory chain etc., was found to be actually mediated by
peroxynitrite.11ꢀ15 Immune cells harness peroxynitrite against cancer cells or other invading pathogens for the
benefit of host.16ꢀ18 However, under pathological conditions such as autoimmune response, ischemicꢀreperfusion
or chronic inflammation, cytotoxicity of peroxynitrite is diverted against the innocent host cells.19ꢀ21
Peroxynitrite unleashes its oxidative capacity and cell damages through multifaceted biochemical
pathways, including twoꢀelectron (2ꢀeꢀ, or polar) or oneꢀelectron (1ꢀeꢀ or radical) oxidation (Figure 1).22
Thermodynamically, OONOꢀ is undoubtedly a strong oxidant, being 36 kCal/mol higher in free energy than its
structural isomer nitrate (NO3 ), due to the presence of a weak OꢀO bond.23 However, OONOꢀ itself is in fact quite
ꢀ
redoxꢀinactive and functions not as an oxidant, but as a proꢀoxidant.19,24ꢀ25 For OONOꢀ to exhibit its oxidative
capacity, a nucleophile would have to attack the terminal oxygen atom, which is negatively charged, to displace
ꢀ
NO2 as the leaving group. However, a high kinetic barrier exists because the negative charge on this terminal
oxygen tends to repel the incoming nucleophile. In fact, OONOꢀ only functions as a nucleophile in biological
matrices and reacts with nothing but electrophiles, including H+, CO2 or various metal ion centers, to yield
HOONO (pKa = 6.8), ONOOCO2 or metalꢀOONOꢀ complexes respectively.26ꢀ36 In sharp contrast to OONOꢀ,
ꢀ
HOONO is a potent electrophilic, readily incorporates a nucleophilic attack to break the weak OꢀO bond and
therefore is a potent 2ꢀeꢀ oxidant.37ꢀ40 HOONO has a reduction potential of +1.4 V (OONOꢀ, 2H+/NO2 , H2O) and
ꢀ
efficiently oxidizes thiols, thioethers and their selenium analogs.37 A fraction of HOONO homolyzes to HO• and
NO2, albeit with a low rate constant.29,41ꢀ42 Homolysis of ONOOCO2 or OONOꢀꢀmetal complexes is more
ꢀ
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efficient in generating radicals, compared to HOONO.43ꢀ50 Depending on cell localizations, the resulting highly
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cytotoxic oneꢀelectron oxidants, i.e. HO•, NO2, and CO3 , may be scavenged by polyenes, vitamin E, BH4, GSH,
•ꢀ
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melatonin, urate or ascorbic acid.51ꢀ58 However, under pathological conditions when cellular redox buffering
capacity is overwhelmed, lipid modification59ꢀ61 via peroxidation/nitration, protein tyrosine nitration62ꢀ69, DNA
oxidation to nucleobase/deoxyribose,70ꢀ72 or DNA strand breakage73ꢀ74 builds up and cell death is often inevitable.
The bioꢀrelevance, elusive mechanism and associated pharmacological opportunities of peroxynitrite in health,
disease and aging have underscored the significance of its detection.75ꢀ76
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Figure 1. Focused peroxynitrite biochemistry.
Fluorescence based techniques are advantageous for biological studies due to the paramount sensitivity,
instrumentational versatility and convenient implementation to complex biological systems. A number of small
molecule fluorescent probes for peroxynitrite have been reported, mainly for in vitro applications. In accordance
with the multiplicity of peroxynitrite biochemistry, the detection mechanisms of these probes are not uniform.
The trifluorocarbonyl (HKGreen1ꢀ3),77ꢀ80 phenyl boronic esters (CBA, or their free acids),81ꢀ84 or vicinal
dicarbonyl85ꢀ86 based probes bear an electrophilic center and directly detect OONOꢀ (Figure 2A). Hydrozine87ꢀ88
and selenium89ꢀ92 based probes detect HOONO (Figure 2B and 2C). The probes (NiSPYs) were designed to
respond to the nitrative capacity of peroxynitrite (Figure 2D).93ꢀ94 In vivo tyrosine nitration occurs via radical
pathway68ꢀ69, but nitration of NiSPy has not been mechanistically elucidated. Some other probes, including DHR
123, APF, HKGreen4, and our PN600, were based on an electronꢀrich aromatic moiety, which may be oxidized
by HOONO, or alternatively by those secondary radicals derived from peroxynitrite (Figure 2E).95ꢀ102
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A. Three peroxynitrite probes targeting at OONO-
N
O
O
-O
CF3
O O
CF3
and their corresponding detection mechanisms.
CF3
F3C
-O
O
O
O
O
-O
O
R
O
O
O
N
O
R
-H+
-H+
-
O
-NO2
O
Cl
Cl
-O
Cl
Cl
COO-
O
O
H2O
OH
O
O
O
O+
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CF3
O
HKGreen-1, R = -CH2COOH
R = -CH2COOH
O
B. Two peroxynitrite probes based on nucleophilic
attack of hydrozine at presumably HOONO
HO
O
O O
O
O
Et2N
O
NEt2
NO2
COO-
O
COO-
O
N
O
N OH
R
-O
N
N
NHR
COO-
O
O
O
O
O
O
O
R = -H
HO
N
HO
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O
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,
H2O
RBPH, R = -Ph
O
NBzF
NO2
NO2
+
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O
NEt2
OH
O
O
+
O
B-
N
O
O
B
OH
-O
O
O
O
O
OH
N
OH
N
R
N
OH
H2O
COOH
O
O
H2O
Me
CBA
Me
Me
O-
Se+
D. A representative peroxynitrite probe aganist the nitrative capacity
C. Representative peroxynitrite probes
HOONO mediated oxidation
O
O
HO
N
derived from OONO-
based on
Se
-H+
of selenoether
OMe
OMe
O2N
Me
Me
Me
Me
Me
Me
Se
O
Me
O
Me
Me
Me
Me
Me
Me
Me
+
N
[ NO2 ]
N
NC
CN
NC
CN
N
N
Me
N
N
N
N
B
Me
B
B
BzSeO-Cy
F
F
Me
Me
Me
F
F
F F
OMe
NiSPY-1
-e-
NO2
-H+
-H+
Se
Me
Me
OMe
N
Me
Me
HN
Me
O
Se
H2N
O
NH2
O
O
X
N
N
Me
Me
Y
H
CyPSe
BOD-Se
COOMe
COOH
E. Representative peroxynitrite probes likely targeting at either HOONO or
secondary reactive radicals.
X+
DHR 123
X=O, or NMe
Y=OMe, OH, NH2, NHMe, or NMe2
X
O
O
X
HO
N
NH
NH
OH
NH2
O
O
O
OH
Et2N
NEt2
O
N
X
X+
X
-e-
-H+
-e-
NH2
PN600
Or
Me
O
R
NH2
NH
NH
Figure 2: Classification of existing peroxynitrite probes by their detection mechanisms.
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A major challenge in developing a peroxynitrite probe lies at potential crossꢀreactivity toward
hypochlorite.96ꢀ97,99ꢀ100,103ꢀ114 We previously discovered that PN600 was able to differentiate peroxynitrite from
hypochlorite via a facile multiꢀchannel ratiometric signal.101 Its differential reactivity toward hypochlorite and
peroxynitrite is herein mechanistically explained. Further, a qualitative guideline was tentatively proposed
regarding the use of an electronꢀrich benzene moiety in detection of hypochlorite and peroxynitrite.
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We note that the term “peroxynitrite” has been conventionally used in a collective fashion in literature to
include both the anionic OONOꢀ and its conjugate acid, peroxynitrous acid (HOONO). Also, damages from
secondary species derived from OONOꢀ, including nitrosoperoxycarbonate (ONOOCO2 ), hydroxyl radical (HO•),
ꢀ
•ꢀ
nitrogen dioxide radical (NO2), and carbonate radical anion (CO3 ) were often generically attributed to
“peroxynitrite”. Throughout the discussions in this manuscript, these conventional and generic uses of
“peroxynitrite” are followed. However, “OONOꢀ” is used in this manuscript when such a generic sense does not
apply, as an effort to avoid ambiguity. The term “hypochlorite” is also used in a collectively sense to include both
HOCl and OClꢀ unless noted.
RESULTS AND DISCUSSIONS
Figure 3: Overall reactivity of PN600 toward peroxynitrite and hypochlorite.
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The overall reaction scheme of PN600 toward peroxynitrite and hypochlorite. In the preceding
communication, PN600 was found to be oxidized to the redꢀemitting 2, via the orangeꢀemitting intermediate 1,
upon addition of peroxynitrite. In comparison, oxidation of PN600 by addition of hypochlorite stopped at 1.
While investigating the oxidation mechanism of PN600 by peroxynitrite and hypochlorite, an additional
fluorescent species, with a maximum excitation wavelength at 380 nm and emission at 450 nm, was also found to
have been generated upon addition of an aliquot of peroxynitrite stock solution, but not hypochlorite (vide infra).
This fluorescent species was determined to be 6ꢀaminoꢀ7ꢀhydroxyꢀ4ꢀmethylcoumarin (3), as its absorption and
fluorescence spectra were identical with those of the independently synthesized compound 3. Therefore, a
revisedꢀoverall reactivity of PN600 toward peroxynitrite and hypochlorite is now summarized in Figure 3.
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Design and synthesis of PN600 analogs. Intuitively, the reactivity of PN600 toward an oxidant is
bestowed by the presence of its electron donating groups, which include a hydroxyl (ꢀOH) and an amino (ꢀNH2)
group. Yet, it is not obvious how the ꢀOH and ꢀNH2 have determined the reactivity of PN600. We envisaged that a
focused structureꢀreactivity relationship should provide mechanistic insights toward PN600 oxidation and
therefore synthesized an additional list of probes, i.e. 4a, 4b, and 4c. Compounds (4aꢀc) were designed by
changing the ꢀOH of PN600 into a less electronꢀdonating hydrogen (ꢀH), methoxy (ꢀOMe) group, or a more
electronꢀdonating ꢀNH2 group respectively. Probes (4aꢀc) were conveniently synthesized in two steps (Figure 4).
Ullmann type condensation between 7ꢀfluoroꢀ4ꢀmethylꢀ6ꢀnitrocoumarin (5) and variously substituted phenols
(6aꢀc) afforded 7aꢀc smoothly without resorting to forcing conditions. Catalytic hydrogenation of 7aꢀc furnished
4aꢀc, the desired analogs of PN600.
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O
O
F
4
5
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7
NO2
Me
O
O
O
R
K2CO3, Cu
5
NO2
Dioxane
Reflux, 6 h
8
9
HO
R
Me
7a
, R = H
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7b, R = -OMe
7c, R = -NO2
6a, R = H
6b, R = -OMe
6c, R = -NO2
H2, 5% Pd/C
MeOH, rt, 6 h
O
O
O
R
NH2
Me
4a, R = H
4b, R = -OMe
4c, R = -NH2
Figure 4. Structures and synthesis of PN600 and a list of analogs 4aꢀc.
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Fluorescence titrations of 4a, 4b, PN600, 4c, by hypochlorite and peroxynitrite. An aliquot of
hypochlorite or peroxynitrite stock was titrated into a solution of probe 4a, 4b, PN600, or 4c (10 ꢀM)
respectively in phosphate buffer (50 mM at pH = 7.4) with 5% DMF as a coꢀsolvent.
All four probes (4a, 4b, PN600 and 4c) exhibited an emission band with a maximum at ca. 535 nm upon
excitation at their absorption maximum of ca. 355 nm. Addition of hypochlorite led to a gradual decrease of their
emissions (Figure 5A, 5B, 5C, 5E). It took ca. 6ꢀ10 equiv. of hypochlorite to consume the emission of probe 4a,
4b, PN600 and 4c (Figure S3). No new fluorescent species were observed to have been generated from oxidation
of 4a and 4b. Oxidation of PN600 by hypochlorite led to accumulation of compound 1 (Figure S4). Further
oxidation of 1 to 2 by hypochlorite was not achieved as the emission of 2 at 595 nm was not observed with
excitation at its maximum absorption wavelength of 550 nm (Figure 5D). Oxidation of 4c by hypochlorite yielded
a redꢀemitting species, with a maximum excitation at ca. 550 nm and emission at 605 nm (Figure 5F). The
spectral properties of this redꢀemitting species are reminiscent of those of compound 2 and we assigned it to 9.
Compound 8 should be the intermediate between 4c and 9.
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2.5x106
2.0x106
1.5x106
1.0x106
B
C
E
A
4b/hypochlorite
4a/hypochlorite
ex = 355 nm
PN600/hypochlorite
4c/hypochlorite
ex = 355 nm
λ
ex = 355 nm
λ
λ
ex = 355 nm
λ
8
5.0x105
9
0.0
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2.5x106
500
600
700
400
500
600
700
Wavelength (nm)
Wavelength (nm)
Wavelength (nm)
Wavelength (nm)
D
F
2.0x106
1.5x106
1.0x106
PN600/Hypochlorite
ex = 550 nm
4c/Hypochlorite
λex = 550 nm
λ
5.0x105
0.0
400
500
600
700
400
500
600
700
Wavelength (nm)
Wavelength (nm)
Figure 5: Fluorescence titrations of probes 4a, 4b, PN600 and 4c by hypochlorite. Excitation was set at 355 nm to
monitor the decrease of the probe emission of 4a (A), 4b (B), PN600 (C) and 4c (E) upon addition of
hypochlorite. Excitation was set at 550 nm to monitor the formation of 2 from PN600 (D), and 9 from 4c (F)
upon addition of hypochlorite.
Figure 6: Proposed oxidation schemes for 4a, 4b, PN600 and 4c by hypochlorite, based on titrations shown in
Figure 5.
Chemical transformations of probes 4aꢀb, PN600 and 4c induced by addition of hypochlorite, with HOCl
presumed to be the active species, are summarized in the Figure 6. The oxidative capacity of HOCl is unleashed
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upon breakage of the weak oxygenꢀchlorine bond. Mechanistically, this occurs when a nucleophile attacks the
electrophilic chlorine atom (ꢀCl) and displaces hydroxide (OHꢀ) as the leaving group. Also based on the fact that
all four probes were effectively oxidized by hypochlorite with similar kinetics (Figure S5), this nucleophile is
most likely ꢀNH2 on the coumarin moiety, which is present in all four probes. Oxidation of the coumarin moiety
led to subsequent formation of an electrophilic Oꢀacylated iminoquinone derivative (as shown in the rounded box
of the Figure 6). The aroxyl moiety of PN600 or 4c, substituted by a strong electron donating group (i.e. –OH in
PN600 or –NH2 in 4c) is nucleophilic enough to furnish a cyclization to give compound 1 and 5 respectively, via
an intramolecular electrophilic aromatic substitution (EAS). In case of 4a or 4b, whose aroxy moiety is not as
nucleophilic, hydrolysis of the acyl oxonium ion presumably competed and no fluorescent products were
observed to have been generated. Inability of hypochlorite to oxidize compound 1 to 2 suggests that the
nucleophilicity of the diarylamino group is not potent enough to initiate an attack at HOCl. In comparison, the
proposed intermediate 8 was oxidized because it has a primary aryl amino group, which is much more
nucleophilic than a diarylamino group.
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3.0x10-5
2.0x10-5
4a
4b
PN600
4c
1.0x10-5
0.0
-0.5
0.0
0.5
Voltage (V)
1.0
1.5
Figure 7. Cyclic voltammograms of probes 4a, 4b, PN600 and 4c in DMF.
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2.5x106
2.0x106
1.5x106
1.0x106
B
E
A
C
Probe 4a
ex=355 nm
Probe 4b
Probe PN600
ex=355 nm
Probe 4c
ex=355 nm
λ
λex=355 nm
λ
λ
7
8
5.0x105
9
0.0
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2.5x106
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700
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500
600
700
Wavelength (nm)
Wavelength (nm)
Wavelength (nm)
Wavelength (nm)
F
D
2.0x106
1.5x106
1.0x106
Probe 4c
λex=550 nm
Probe PN600
ex=550 nm
λ
5.0x105
0.0
400
500
600
700
400
500
600
700
Wavelength (nm)
Wavelength (nm)
Figure 8. Fluorescence titrations of probes 4a, 4b, PN600 and 4c by peroxynitrite. Excitation was set at 355 nm to
monitor the decrease of the probe emission of 4a (A), 4b (B), PN600 (C) and 4c (E) upon addition of
peroxynitrite. Excitation was set at 550 nm to monitor the formation of 2 from PN600 (D) and 9 from 4c (F) upon
addition of peroxynitrite.
Figure 9. Oxidations of probe 4a, 4b, PN600 and 4c, which were mediated by peroxynitrite, but not hypochlorite.
The first oxidative potential of all four probes (4aꢀc and PN600) was essentially identical at ca. +0.8 V as
determined by cyclic voltammetry despite the different nature of the substituent on the aroxy moiety of 4aꢀc and
PN600 (Figure 7), suggesting that the coumarin moiety is where oxidation occurs most readily in each probe.
Also, the first oxidative potential of 1 was measured to be ꢀ0.14 V.101(SI) So, probe 4aꢀc or PN600, of a higher
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oxidative potential, was oxidized by hypochlorite, while 1, of a lower oxidative potential, was not. This
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interesting observation clearly indicates that oxidation by hypochlorite is not under thermodynamic control.
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All the transformations of probes 4aꢀb PN600 and 4c induced by hypochlorite (Figure 5) were also
observed to have occurred upon addition of peroxynitrite (Figure 8, gray). This is not surprising since HOONO
can presumably mediate 2ꢀeꢀ oxidation, analogously to HOCl. However, we also noticed that peroxynitrite
mediated some transformations, which were not seen with hypochlorite. The first such transformation was the
formation of the redꢀemitting compound 2 from further oxidation of the intermediate 1 (Figure 8D and 9, red).
Second, an emission band at 450 nm, of compound 3, appeared upon peroxynitrite mediated oxidation of all four
probes (Figure 8A, 8B, 8C, 8E and 9, blue).
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•ꢀ
•
Peroxynitrite can mediate 1ꢀeꢀ oxidations through various secondary radicals including OH•, CO3 , NO2
etc, while hypochlorite cannot. So, these transformations unique to peroxynitrite have presumably occurred via a
radical mechanism. Compound 1 contains a paraꢀaminophenol moiety and such electron rich polyaromatics are
known radical scavengers, i.e. to be readily oxidized by radicals.
Formation of 3 was rather unexpected because this necessitates oxidation of the less electronꢀrich aroxy
moiety in the presence of the more electronꢀrich coumarin moiety. However, this seemingly unexpected
observation is in fact reasonable. Secondary radicals from peroxynitrite are highly oxidative. Thus, they can
indiscriminately abstract an electron from whichever oxidizable substrate they encounter first. In other words,
oxidation by these radicals is kinetically controlled and steric accessibility, rather than oxidative potential,
determines the profile of the reaction outcome. It happened that the coumarin moiety of probes 4aꢀc and PN600 is
sterically shielded by the aroxy moiety. This explains why a kinetic oxidation of the less electronꢀrich aroxy
moiety occurred, and ultimately resulted in the formation of 3.
Mechanistic explanation to the overall reactivity of PN600 toward hypochlorite and peroxynitrite.
With the mechanistic insights from these titrations, reactivity of PN600 toward hypochlorite and peroxynitrite are
well accounted for and summarized in the Figure 10. Nucleophilic attack commencing from its –NH2 at HOCl or
HOONO followed by a condensation to displace a chloride (ꢀClꢀ) or a hydroxide (ꢀOHꢀ) respectively, yields an
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acyl oxonium species (10). Alternatively, this process can potentially take place via a radical pathway and be
mediated by peroxynitrite. The resulting acyl oxonium ion (10) undergoes a ring closure to afford the
intermediate 1. This has explained why the addition of either hypochlorite or peroxynitrite can induce the
formation of compound 1. However, lack of a nucleophile on the scaffold of 1 strong enough to attack HOCl
renders it stable toward hypochlorite. In comparison, the secondary radicals from peroxynitrite readily oxidize
compound 1 to 2. Kinetic radical oxidation to the 3ꢀhyroxyphenoxyl moiety can occur, in a fashion similar to the
stepꢀwise radical oxidation of phenol to quinone. Ultimately, the diaryl ether linkage breaks upon hydrolysis to
generate 3.
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Figure 10. A proposed mechanistic explanation to the observed reactivity of PN600 toward both hypochlorite and
peroxynitrite. Note: Magenta arrows highlight the radical reactions.
An empirical guideline for development of probes targeting at peroxynitrite or hypochlorite. With
the few probes of this manuscript and rich literature work, in particular those from Yang et al. and Nagano et al.,
we were able to categorize various electronꢀrich aromatic structures based on their reported reactivity toward
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peroxynitrite and hypochlorite (Chart 1). Tentatively, this summary may serve as a qualitative guideline for future
use of electronꢀrich aromatic rings as the recognition moiety of a fluorescent probe for either hypochlorite or
peroxynitrite detection:
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1. A phenolic hydroxyl (ArꢀOH) is not nucleophilic enough to attack HOCl and initiates an oxidation.97,101ꢀ
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102,115ꢀ117
2. A diarylamino (ArꢀNHꢀAr’) group does not react with HOCl.101ꢀ112,115
3. A monoarylamino (ArꢀNH2) group with a paraꢀmethine (ꢀCHRR’, where R and R’ are alkyl or aryl) group
is mildly reactive toward HOCl.98 Removal of the paraꢀmethine reduces this reactivity.
4. A monoarylamino (ArꢀNH2) group with an oxygen or nitrogen attached to its para position is reactive
toward HOCl.96ꢀ97,100,102,116 A broad spectrum of substitutions to this oxygen or nitrogen atom seems to have
been tolerated, including alkyl, aryl or acyl. Further substitution of this monoarylamino group by two small
alkyl groups into monoaryldialkyl amino groups (ArꢀNMe2, or ArꢀNEt2) does not seem to have greatly
suppressed its reactivity toward HOCl.
5. A phenolate (ArꢀOꢀ) is reactive toward HOCl.118ꢀ119
6. All aforementioned structures are expected to exhibit reactivity toward peroxynitrite. Reaction mechanism
can be 2ꢀeꢀ or 1ꢀeꢀ depending on the existence of a nucleophilic amino group or not.
We judiciously listed phenolate as a separate category because of HKOCl-128a, which seems not to agree
with the rule 1. However, this inconsistency can be rationalized. A phenol is an acidic functional group with a pKa
of ca. 10. At physiological pH, more than 99% of a phenol should exist in form of its conjugate acid accordingly
to HendersonꢀHasselbach equation. However, the electronꢀwithdrawing nature of the BODIPY core within the
scaffold of HKOCl-1 should also be considered. It might have lowered the pKa of the ortho phenolic OH group,
as a combined result of the inductive effect and longꢀrange electronic interaction120ꢀ121, to allow a significant
portion of HKOCl-1 stay in form of the corresponding phenolate at physiological pH. The phenolate is intuitively
much more nucleophilic than the corresponding phenol and expected to be able to attack HOCl and initiate a 2ꢀeꢀ
oxidation. Though, the exact pKa value of HKOCl-1 was not reported, this analysis was supported by the recent
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report, from the same authors, that orthoꢀsubstitution to the phenol by electronꢀwithdrawing halogen atoms, i.e. –
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Cl or –F respectively, further improved the probe reactivity toward hypochlorite.28b
7
Development of hypochlorite specific probe without interferences from peroxynitrite will be challenging.
An electronꢀrich aromatic substrate may survive HOONO, but not those secondary radicals from peroxynitrite. In
fact, even notꢀsoꢀelectronꢀrich substrates do not survive these radicals, including umbelliferone, salicylate, and
terephthalate.25d In other words, electronꢀrich aromatics are better suited for unspecific detection of hypochlorite
and peroxynitrite, or specific detection of peroxynitrite over hypochlorite.
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Category I
Category II
Category III
Category IV
Category V
Note: 1) In ꢀꢀ colored boxes are probes reactive toward secondary radicals from
peroxynitrite only; 2) In ꢀꢀ are those reactive toward not only those secondary
radicals from peroxynitrite, but also HOCl and HOONO.
Chart 1:Classification of probes for hypochlorite and peroxynitrite based on oxidation of an electron-rich
aromatic ring.
An embodiment of the guideline. Following this guideline, we designed and synthesized 11 (Figure 11)
as a selective peroxynitrite probe. It falls within the category III and is expected to display good reactivity toward
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peroxynitrite, but poor reactivity toward hypochlorite. A close analog (12, Figure 11) of an unnamed probe of
category IV, which reacted well with both peroxynitrite and hypochlorite, was synthesized (Figuer 12) and titrated
sideꢀbyꢀside with 11 (Figure 13).
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Figure 11. Oxidation pathways of probes 11, 12 and 14 by peroxynitrite.
Figure 12. Synthetic Schemes of probes 11 and 14.
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Compound 11 and 12 make a good comparison as their structures differ from each other only by the
position of –NH2. Yet, this seemingly minor variation makes a huge difference with respect to their reactivity
toward peroxynitrite and hypochlorite.
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Synthesis of probes 11 and 14 started from condensation of 16 and 17 under Ullmann conditions to afford
the diaryl ether 18 in a moderate yield. Demethylation of 18 in refluxing 48% HBr gave 19 nearly quantitatively.
Condensation of 19 with 20 or 22 in 70% H2SO4 at high temperature yielded the desired coumarin (21) or Rhodol
derivative (23). Reduction of the nitro group on 21 or 23 with SnCl2 by refluxing in concentration HCl furnished
probe 11 or 14 in good yields. Catalytic hydrogenation of 23 also partially reduced the Rhodol scaffold and
therefore not suitable for preparation of 14.
Probe 11 and 12 (10 ꢀM) in phosphate buffer (50 mM, at pH=7.4) containing 5% DMF were titrated by
hypochlorite and peroxynitrite (Figure 13A and 13B). Emission of their oxidation product, 7ꢀhydroxyꢀ4ꢀ
methylcoumarin (13), with a maximum excitation at 360 nm and an emission at 450 nm, was monitored. The
reported high reactivity of probe 12 toward hypochlorite was verified. Addition of ca. 6 equiv. of hypochlorite led
to a near saturation of fluorescence enhancement. Probe 11 was not totally inert to hypochlorite. However, its
signal enhancement was less than 2% of that from probe 12. Addition of a higher dose of hypochlorite than 6
equiv. led to a gradual bleaching of fluorescence. Two probes (11 and 12) yielded an emission of comparable
intensity upon addition of peroxynitrite. Only, it took ca. 10 equiv. of peroxynitrite to saturate the signal
enhancement of probe 12, while roughly twice as much of peroxynitrite was necessary to saturate the signal
enhancement of probe 11. The spectral titrations of this pair of probes exemplified the viability of the
aforementioned guideline.
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1.0x107
7.5x106
5.0x106
2.5x106
A
λ
ex=360 nm
em=450 nm
λ
Hypochlorite Induced
signal enhancement by:
Probe 11
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Probe 12
0.0
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Hypochlorite Equivalence
4.0x106
B
Peroxynitrite induced
signal enhancement by:
Probe 11
3.0x106
2.0x106
λ
λ
ex=360 nm
em=450 nm
Probe 12
1.0x106
0.0
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Peroxynitrite Equivalence
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Figure 13. Dose dependent fluorescence enhancement of probes 11 and 12 by hypochlorite (A) and peroxynitrite
(B).
2.5x105
Peroxynitrite induced signal
enhancement by Probe 14.
2.0x105
λ
λ
ex=520 nm
em=550 nm
1.5x105
1.0x105
5.0x104
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Peroxynitrite Equivalence
Figure 14. Dose dependent fluorescence enhancement of probes 14 by peroxynitrite.
By attaching the 3ꢀaminophenyl group to the oxygen atom of a rhodol fluorophore (15), a peroxynitrite
probe (14) of longer wavelength, was designed (Figure 11) and synthesized (Figure 12). Probe 14 inherited the
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reactivity of probe 11 toward peroxynitrite (Figure 14). Probe 14 shows no reactivity toward large excess of H2O2,
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KO2, NO, O2 (Figure S13). Addition excess hypochlorite turned on the emission at 550 nm by ca. 5 fold, while
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excess peroxynitrite enhanced the emission at 550 nm by over 45 fold (Figure S12). This proved that probe 14 is
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selective toward peroxynitrite over hypochlorite.
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CONCLUSION
PN600 is an interesting fluorescent probe, which yields an unusual fourꢀchannel fluorescence signal upon
oxidation by peroxynitrite. It showed crossꢀreactivity toward hypochlorite. Oxidation by hypochlorite could be
spectroscopically differentiated from oxidation by peroxynitrite. Through combined use of spectral titrations,
electrochemical studies and fundamental organic arrowꢀpushing mechanisms, we were able to satisfyingly
explain the observed reaction profile of PN600 toward peroxynitrite and hypochlorite. With findings of this work
and previous literature publications, we have proposed a qualitative guideline for use of electronꢀrich aromatic
rings as the reactive moiety in constructing fluorescent probes for hypochlorite or peroxynitrite. Following this
guideline, we further developed two peroxynitrite probes, which are indeed only minimally reactive toward
hypochlorite. This supports the viability of this guideline.
EXPERIMENTAL
1
All chemicals and solvents were analytical grades and used without further purification. The HꢀNMR and
13CꢀNMR spectra were acquired on a Bruker AVꢀ400 spectrometer. Chemicals shifts were referenced to the
residue solvent peaks and listed in ppm. Coupling constants were given in Hz. ESIꢀHRMS was acquired on a
TOF mass spectrometer and EIꢀHRMS on a Micromass GCT spectrometer. UVꢀVis absorption spectra were
acquired on a SHIMADZU UVꢀ2600 UVꢀvis spectrophotometer. Fluorescence emission spectra were acquired on
a PTIꢀQM4 steadyꢀstead fluorimeter equipped with a 75 Watt Xenon arcꢀlamp and a R928 PMT. All excitation
spectra were corrected. All emission spectra were not corrected with respect to the PMT sensitivity at different
wavelengths. The excitation and emission slits were both 2 nm for all experiments. Fluorescence titrations were
carried out by addition of an aliquot of the stock solution of hypochlorite, peroxynitrite or other species.
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Electrochemical analyses were performed by using a computer controlled CHI 660c electrochemical station
(Chenhua Co. Ltd, Shanghai, China). The electrochemical experiments were carried out in a threeꢀelectrode
electrochemical cell under nitrogen atmosphere. Working electrodes were 3ꢀmm diameter glassy carbon (GC)
disks, used in conjunction with a Pt auxiliary electrode and an Ag/AgCl wire reference electrode. The
concentration of each sample was 1mM, and was dissolved in N,NꢀDimethylformamide (DMF).
Tetrabutylammonium Perchlorate (TBAP) acted as supporting electrolyte and its concentration was 0.1 M. The
data was obtained from the first cycle in cyclic voltammetry (CV), and the scan rate was 100 mV/s.
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6-Amino-4-methyl-7-phenoxy-2H-chromen-2-one (4a). Mix 7a (50mg, 0.17 mmol), EtOH (5 mL) and
concentrated HCl (5 mL) in a 25 mL round bottom flask. Add SnCl2•2H2O (198 mg, 0.88 mmol) into the flask
o
slowly with constant stirring. Heat the reaction mixture to 40 C for 6 hrs before cooling to room temperature.
The reaction mixture was poured in concentration NaHCO3 solution and extracted with CH2Cl2. The organic layer
was dried with MgSO4, filtered, passed through a silica plug to remove the colored impurities and evaporated to
dryness under reduced pressure. The crude product was recrystallized in CH2Cl2 to afford 4a as a yellow solid (30
mg, 67%). 1H NMR(400 MHz, CDCl3): δ(ppm) 7.39 (t, J = 8.0 Hz, 2H) , 7.20 (t, J = 8.0 Hz, 1H), 7.06 (d, J = 8.0
Hz, 2H), 6.94 (s, 1H), 6.69 (s, 1H), 6.16 (s, 1H), 3.96 (s, 2H), 2.37 (s, 3H); 13C NMR (100 MHz, CDCl3): δ(ppm)
161.3, 155.3, 151.8, 148.3, 147.2, 134.5, 130.1, 124.7, 119.3, 115.6, 113.3, 109.2, 105.4, 18.8; EIꢀHRMS (m/z)
[M+] calcd. for C16H13NO3, 267.0895; found 267.0893.
6-Amino-7-(3-aminophenoxy)-4-methyl-2H-chromen-2-one (4c). Mixture of 7c (140 mg, 0.41 mmol), 5%
Pd/C (a spatula end) and 10 mL MeOH was stirred in an H2 atmosphere (0.4 MPa) in a pressure vessel for 4 hrs at
room temperature before the system was vented. The mixture was passed through a silica plug and evaporated to
dryness under reduced pressure to afford a viscous residue. Upon recrystallization in a mixed solvent of CH2Cl2
and petroleum ether, 4c was obtained as a light yellow solid (102 mg, 88%). 1HꢀNMR(400 MHz, CDCl3): δ(ppm)
7.16 (t, J = 8.0 Hz, 1H) , 6.95 (s, 1H), 6.80 (s, 1H), 6.52 (dd, J = 8.0,1.6 Hz, 1H), 6.45 (dd, J = 8.0, 1.6 Hz, 1H),
13
6.39 (s, 1H), 6.20 (s, 1H), 2.39 (s, 3H); C NMR (100 MHz, CDCl3): δ(ppm) 161.4, 156.5, 151.9, 148.3, 148.2,
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147.2, 134.6, 130.7, 115.6, 113.3, 111.4, 109.1, 108.9, 105.9, 105.7, 18.8; ESꢀHRMS (m/z): [M]+ calcd. for
C16H14NO3, 283.1077; found 283.1080.
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4-Methyl-6-nitro-7-phenoxy-2H-chromen-2-one (7a). Mix 5 (50 mg, 0.22 mmol), 6 (23 mg, 0.25 mmol),
K2CO3 (34 mg, 0.25 mmol), fresh Cu (5 mg) and 1,4ꢀdioxane (20 mL) in a 50 mL round bottom flask. The
mixture was heated to reflux overnight with stirring before cooled and filtered through a celite pad. The filtrate
was dried under reduced pressure and chromatographed with petroleum ether/EtOAc (15:4, v/v) to obtain 7a as a
light yellow solid (35 mg, 53%). 1H NMR(400 MHz, CDCl3): δ(ppm) 8.29 (s, 1H) , 7.47 (t, J = 8.0 Hz, 2H), 7.31
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(t, J = 8.0 Hz, 1H), 7.13 (d, J = 8.0 Hz, 2H), 6.77 (s, 1H), 6.28 (s, 1H), 2.47 (s, 3H); C NMR (100 MHz, CDCl3)
δ(ppm) 158.9, 156.8, 154.7, 153.9, 151.1, 136.5, 130.5, 126.2, 123.1, 120.4, 114.7, 114.5, 106.4, 18.6; EIꢀMS
(m/z) [M]+ calcd. for C16H11NO5, 297.0637; found 297.0638.
4-Methyl-6-nitro-7-(3-nitrophenoxy)-2H-chromen-2-one (7c). Mix 5 (500 mg, 2.24 mmol), 6c (311 mg, 2.24
mmol), K2CO3 (310 mg, 2.25 mmol), fresh Cu powder (40 mg) and 1,4ꢀdioxane (35 mL) in a 50 mL round
bottom flask. The mixture was heated to reflux overnight with stirring before cooled and filtered through a celite
pad. The filtrate was dried under reduced pressure and chromatographed with EtOAc to obtain 7c as a yellow
1
solid (440 mg, 57%). H NMR(400 MHz, CDCl3): δ(ppm) 8.36 (s, 1H) , 8.14 (d, J = 8.0 Hz, 1H), 7.90 (s, 1H),
7.64 (t, J = 8.4 Hz, 1H), 7.47 (dd, J = 8.4, 2.0 Hz, 1H), 6.93 (s, 1H), 6.37 (s, 1H), 2.51 (s, 3H); 13C NMR (100
MHz, CDCl3): δ(ppm) 158.4, 157.0, 155.7, 152.1, 150.9, 149.5, 137.4, 131.2, 125.3, 123.6, 120.2, 116.5, 115.8,
114.1, 108.69, 18.7; ESIꢀHRMS (m/z): [M+H]+ calcd. for C16H11N2O7, 343.0561; found 343.0553.
7-(3-Aminophenoxy)-4-methyl-2H-chromen-2-one (11). Mix 21 (174 mg, 0.585 mmol), EtOH (10 mL) and
concentrated HCl (10mL) in a 50 mL round bottom flask. Add SnCl2•2H2O (700 mg, 3.10 mmol) into the flask
slowly with constant stirring. Heat the mixture to 40 oC for 6 hr before cooling to room temperature. The reaction
mixture was poured in concentration NaHCO3 solution and extracted with CH2Cl2. The organic layer was dried
with MgSO4, filtered, passed through a silica plug to remove colored impurities and evaporated to dryness under
reduced pressure. The crude product was recrystallized in a mixed solvent of CH2Cl2 and petroleum ether to
1
afford 4a as a light yellow solid (142 mg, 91 %). HꢀNMR(400 MHz, CDCl3) δ(ppm) 7.52 (d, J = 8.4 Hz, 1H) ,
7.15 (t, J = 8.0 Hz, 1H), 6.94 (dd, J = 8.0, 1.2 Hz, 1H), 6.89 (s, 1H), 6.52 (d, J = 8.0 Hz, 1H), 6.44 (d, J = 8.0 Hz,
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1H), 6.39 (s, 1H), 6.17 (s, 1H), 3.75 (s, 2H), 2.41 (s, 3H); 13C NMR (100 MHz, CDCl3) δ(ppm) 161.1, 160.9,
156.2, 154.9, 152.2, 148.3, 130.7, 125.6, 115.0, 114.3, 112.8, 111.6, 109.9, 106.7, 105.4, 18.7; ESIꢀMS (m/z)
[M+H]+ calcd for C16H14NO3, 268.0968; found 268.0982．
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3'-(3-Aminophenoxy)-6'-(diethylamino)-3H-spiro[isobenzofuran-1,9'-xanthen]-3-one (14). Mix 23 (160 mg,
0.314 mmol), EtOH (10 mL) and concentrated HCl (10mL) in a 50 mL round bottom flask. Add SnCl2•2H2O
o
(350 mg, 1.55 mmol) into the flask slowly with constant stirring. Heat the mixture to 60 C for 6 hr before
cooling to room temperature. The reaction mixture was poured in concentration NaHCO3 solution and extracted
with CH2Cl2. The organic layer was dried with MgSO4, filtered, and evaporated to dryness under reduced
pressure. The crude product was chromatographed with CH2Cl2/MeOH (50:1, v/v) to afford 14 as a light pink
solid (52 mg, 34.6%). 1H NMR(400 MHz, CDCl3) δ(ppm) 8.00 (d, J = 7.6 Hz, 1H) , 7.86 (t, J = 7.2 Hz, 1H), 7.60
(t, J = 7.6 Hz, 1H), 7.21 (d, J = 7.6 Hz, 1H), 7.12 (t, J = 8.0 Hz, 1H), 6.83 (d, J = 2.4 Hz, 1H), 6.65~6.71 (m, 2H),
6.57 (d, J = 8.8 Hz, 1H), 6.42~6.48 (m, 3H), 6.37 (t, J = 2.4 Hz, 1H), 6.34 (d, J = 2.8 Hz, 1H), 3.75 (s, 2H), 3.35
(q, J = 6.8 Hz, 4H), 1.16 (t, J = 6.8 Hz, 6H); 13C NMR (100 MHz, CDCl3) δ(ppm) 172.1, 161.9, 159.6, 155.6,
155.4, 155.3, 152.2, 150.8, 137.3, 133.1, 132.1, 131.8, 131.4, 129.9, 127.4, 126.6, 116.5, 116.4, 113.6, 112.3,
111.0, 108.9, 108.3, 107.6, 100.1, 86.8, 47.0, 15.1; EIꢀMS (m/z) [M+] calculated for C30H26N2O4, 478.1893;
found 478.1898.
4-Methyl-7-(3-nitrophenoxy)-2H-chromen-2-one (21). Mix 19 (700 mg, 3.03 mmol) and 75% H2SO4 (20 mL)
in a 50 mL round bottom flas. Add 20 (433 mg, 3.33 mmol) dropwise and let the reaction sit with vigorous
stirring at room temperature for 24 hrs. Pour the reaction mixture into crushed ice and collect the solid via suction
filtration. The crude product was chromatographed with CH2Cl2/Petroleum ether (5:2, v/v) to afford 21 as a light
yellow solid (340 mg, 37.8%). 1H NMR(400 MHz, CDCl3) δ(ppm) 8.06 (dd, J = 8.4, 1.2 Hz, 1H) , 7.89 (t, J = 2.4
Hz, 1H), 7.62 (d, J = 8.4 Hz, 2H), 7.58 (t, J = 8.4 Hz, 1H), 7.42 (dd, J = 8.4, 2.4 Hz, 1H), 6.99 (dd, J = 8.4, 2.4
Hz, 1H), 6.96 (d, J = 8.4 Hz, 1H), 6.25 (d, J = 1.2 Hz, 1H), 2.44 (d, J = 1.2 Hz, 3H); 13C NMR(100 MHz,
CDCl3) δ(ppm) 160.4, 158.9, 156.6, 155.0, 151.9, 130.8, 126.3, 125.5, 119.2, 116.5, 114.9, 114.3, 113.8, 106.8,
18.7; ESIꢀMS (m/z) [M+H]+ calculated for C16H12NO5, 298.0710, found 298.0719.
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3'-(Diethylamino)-6'-(3-nitrophenoxy)-3H-spiro[isobenzofuran-1,9'-xanthen]-3-one (23). Mix 19 (174 mg,
0.585 mmol) and 22 (235 mg, 0.585 mmol) and 85% H3PO4 (15 mL) in a 25 mL round bottom flask. Heat the
flask to 100 oC with stirring for 5 hrs. Pour the mixture into saturated NaHCO3 solution and extract with CH2Cl2.
The organic layer was dried with MgSO4, filtered, and evaporated to dryness under reduced pressure. The crude
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product was chromatographed with CH2Cl2:MeOH (100:1) to afford 23 as a pink solid (310 mg, 81%). H
NMR(400 MHz, CDCl3) δ(ppm) 8.00~8.03 (m, 2H) , 7.90 (t, J = 4.0 Hz, 1H), 7.69 (t, J = 8.0 Hz, 1H), 7.62 (t, J =
8.0 Hz, 1H), 7.53 (t, J = 8.0 Hz, 1H), 7.39 (dd, J = 8.0, 4.0 Hz, 1H), 7.25 (d, J = 8.0 Hz, 1H), 6.88 (d, J = 2.4 Hz,
1H), 6.79 (d, J = 8.8 Hz, 1H), 6.69 (dd, J = 8.0, 2.4 Hz, 1H), 6.58 (d, J = 8.0 Hz, 1H), 6.43 (d, J = 2.8 Hz, 1H),
6.37 (dd, J = 8.8, 2.8 Hz, 1H), 3.36 (q, J = 7.2 Hz, 4H), 1.17 (t, J = 7.2 Hz, 6H); 13C NMR (100 MHz, CDCl3)
δ(ppm) 169.6, 157.6, 157.4, 153.2, 153.0, 152.9, 149.8, 135.1, 130.7, 130.1, 129.8, 129.0, 127.3, 125.3, 125.1,
124.2, 118.8, 115.9, 114.5, 114.3, 108.8, 107.1, 105.0, 97.7, 44.7, 27.1, 12.6; ESIꢀMS (m/z); [M+H]+ calc. for
C30H25N2O6, 509.1707; found 509.1716.
Supporting Information.
Additional fluorescence spectral results, NMR and HRMS spectra of new compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.”
Funding Sources
The work is supported by Shanghai RisingꢀStar Program (No. 13QA1401200), Doctoral Fund of Ministry of
Education of China (No. 20110074120008) and National Natural Science Foundation of China (Nos. 21106043
and 21372080).
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