A significant improvement of the efficacy of radical oxidant probes by the kinetic isotope effect

Article abstract of DOI:10.1002/anie.201002228

A breath of fresh air: The rate of aerial oxidation of dihydroethidium and hydrocyanine radical oxidant probes can be selectively reduced by deuteration (see picture). The reaction rate between the deuterated compounds and the superoxide radical was reduced by a much smaller factor because of mechanistic differences between the two reactions.

Full text of DOI:10.1002/anie.201002228

Communications
DOI: 10.1002/anie.201002228
Fluorescent Probes
A Significant Improvement of the Efficacy of Radical Oxidant Probes
by the Kinetic Isotope Effect**
Kousik Kundu, Sarah F. Knight, Seungjun Lee, W. Robert Taylor, and Niren Murthy*
The accurate and sensitive detection of radical oxidants is a
central problem in the field of chemical biology.[1] Radical
oxidants can be detected in vitro with fluorescent leucodyes
such as dihydroethidium (DHE),[2] dihydrorhodamine
(DHR),[3] the hydrocyanines,[4] redoxfluor-1,[5a] and their
organelle-specific analogues.[5b] Although these probes are
widely used in cell culture, their accuracy is compromised by
their high levels of background fluorescence,[6] which is
caused by their spontaneous oxidation that is catalyzed by
light or oxygen. Radical oxidants can be detected by using
DHE, DHR, and the hydrocyanines, as they undergo an
amine oxidation reaction[7] with cellular oxidants, such as
superoxide or hydroxyl radicals. However, the probes also
generate background fluorescence by undergoing the same
amine oxidation reaction with air and light; this reaction is
often attributed to the effects of singlet oxygen (1O2).[8]
The mechanism of the amine oxidation differs signifi-
cantly for reactions that involve either radical oxidants or
singlet oxygen, and in particular, cleavage of the a-amine
transfer (ET)/proton transfer (PT) mechanism or a direct
hydrogen atom transfer (HAT) mechanism, and generally has
a lower KIE than oxidation with singlet oxygen.[10] For
example, the KIE for the oxidation of N,N-dime-
thyl([D2]benzyl)amine with the tert-butoxy radical (tBuOC)
is only 1.4 Æ 0.7,[10a–c] and the KIE for the mechanistically
similar oxidation of N,N-dimethyl([D2]benzyl)aniline with
cytochrome P450 is 1.8 Æ 0.2.[10a] This difference in KIEs[11] for
the amine oxidation reaction between singlet oxygen and
radical oxidants offers the possibility of selectively slowing
down the aerial oxidation of radical oxidant probes while
maintaining their reactivity with cellular radical oxidants.
Herein we demonstrate that the efficacy of the commonly
used radical oxidant probes, DHE (1), H-Cy3 (3), H-Cy5 (5),
H-Cy7 (7), and DHR (9), can be dramatically improved by
À
deuteration at their a-amine C H bond (Figure 1). Deuter-
ated analogues of DHE (1), H-Cy3 (3), H-Cy5 (5), and H-Cy7
(7) have large KIEs (3.7–4.7) for aerial oxidation; however,
their KIEs for oxidation with the superoxide radical anion are
only between 2.5–2.8. This difference in KIEs causes the
deuterated radical oxidant probes to generate less back-
ground fluorescence, but to still generate similar levels of
fluorescence in cells that are stimulated to produce radical
oxidants. Deuterated radical oxidant probes were signifi-
cantly more accurate than their hydrogen analogues in the
detection of radical oxidants in vitro, in cell culture, and
in vivo. For example, the deuterated DHE analogue DDE
had several advantages over its hydrogen analogue because of
its lower background fluorescence. In particular, DDE had
greater storage stability and higher accuracy than DHE, and
was also used to detect radical oxidants produced in cell
culture from angiotensin II (Ang II) stimulated rat aortic
smooth muscle cells (RASM), whereas the oxidants were not
detected by using DHE under identical experimental con-
ditions. Similarly, D-Cy7 was also significantly better than its
hydrogen analogue H-Cy7 (7) in the detection of radical
oxidants in vivo because of its low background fluorescence.
Based on these results, we anticipate numerous applications
of deuterated radical oxidant probes in biology and medicine.
Deuterated analogues of DHE (1), H-Cy3 (3), H-Cy5 (5),
H-Cy7 (7), and DHR (9) were synthesized in excellent yields
(> 93%) by reduction of commercially available ethidium
bromide (11), Cy3 (12), Cy5 (13), Cy7 (14), and rhodamine
(15) dyes, respectively, with sodium borodeuteride (see the
Supporting Information). This reduction procedure specifi-
cally introduces a deuterium atom at the a-amine carbon
atom of ethidium and the cyanines, and at the e-amine carbon
atom of rhodamine. We investigated the stability of the
deuterated probes 2, 4, 6, 8, and 10 to deuterium/hydrogen
(D/H) exchange, and found them to be resistant to D/H
À
C H bond occurs at different points along the reaction
coordinate for oxidation with these two oxidants. For
example, amine oxidation by singlet oxygen proceeds via an
À
exciplex intermediate, in which the C H bond cleavage
occurs in the rate-determining step. This oxidation reaction
therefore exhibits a relatively high kinetic isotope effect
(KIE).[9] For instance, the KIE for the oxidation of N,N-
dimethyl([D2]benzyl)amine with singlet oxygen is 3.06 Æ
0.06.[8a] In contrast, a radical mechanism for amine oxidation
proceeds through a sequence that involves either an electron
[*] K. Kundu, S. Lee, Prof. W. R. Taylor, Prof. N. Murthy
The Wallace H. Coulter Department of Biomedical Engineering and
Parker H. Petit Institute of Bioengineering and Biosciences
Georgia Institute of Technology
Atlanta, GA 30332 (USA)
Fax: (+1)404-894-4243
E-mail: niren.murthy@bme.gatech.edu
S. F. Knight, Prof. W. R. Taylor
Cardiology Division, Department of Medicine
Emory University School of Medicine
Atlanta, GA 30322 (USA)
Prof. W. R. Taylor
Cardiology Division, Atlanta VA Medical Center
Decatur, GA 30032 (USA)
[**] This work was supported by the Georgia Tech/Emory Center for the
Engineering of Living Tissues (funded by NSF-EEC-9731643)
(N.M.), NSF-BES-0546962 Career Award (N.M.), NIH UO1
HL80711-01 (N.M.), NIH R21 EB006418 (N.M.) NIH RO1
HL096796-01 (N.M.), NIH RO1 HL090584 (W.R.T.) and J&J/GT
Health Care Innovation Seed Grant Proposal (N.M.).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201002228.
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analogues were incubated
with SOD and xanthine/
xanthine oxidase, and the
rates of oxidation of the
probes 1–10 were measured
by fluorescence spectrosco-
py. Table 1 shows that the
deuterated radical oxidant
probes 2, 4, 6, 8, and 10
reacted with superoxide
radicals
1.7–2.8
times
slower than their hydrogen
analogues. Importantly, the
KIEs observed for oxida-
tion of 2, 4, 6, 8, and 10 with
superoxide radicals are
lower than their corre-
sponding KIEs for aerial
oxidation (1.7–2.8 versus
3.7–4.7). The lower KIEs
observed for oxidation
with superoxide radicals
suggests that the oxidation
mechanism for these dyes
with superoxide radicals is
different than for aerial oxi-
dation. In summary, deuter-
ated radical oxidant probes
are stabilized with respect
to aerial oxidation, but still
maintain a high reactivity
toward cellular oxidants.
Figure 1. a) Nonfluorescent tertiary amines 1–10 that are oxidized to fluorescent iminium cations 11–15 by a
radical-mediated amine oxidation (bold arrow) are probes for radical oxidants. However, they also generate
background fluorescence by aerial oxidation (dotted arrow, mediated by 1O2). b) Selective suppression of
background fluorescence for the deuterated probes leads to a higher signal to noise (S/N) ratio in cell culture
and in vivo.
exchange under aqueous conditions (see the Supporting
Information).
DHE is currently the most commonly used radical oxidant
probe in biology and has been used extensively in cell culture
studies.[2] However, its spontaneous aerial oxidation generates
high background fluorescence that limits its accuracy, and also
makes its storage and handling a challenge. Therefore, we
investigated if deuteration would improve the accuracy and
storage life of DHE. We compared the performances of DHE
and DDE in detecting the hydroxyl radical in vitro at
nanomolar concentrations (see the Supporting Information).
Figure 2a shows that DDE has greater accuracy than DHE
for the detection of the hydroxyl radical at nanomolar
concentrations. For example, DDE could be used to detect
nanomolar levels of radical oxidants and had an R2 (linear
regression) value of 0.97 in this concentration range. In
contrast, we were unable to detect radical oxidants with
precision using DHE in the same concentration range. This
result is shown by an R2 value of 0.79, and also a much higher
standard deviation per measurement than the data for DDE.
We further investigated if deuteration would improve the
storage life of DHE. Figure 2b shows that the storage profile
of DDE is significantly better than that of DHE. For example,
60% of solid DHE was oxidized after 10 days storage,
whereas only 20% of solid DDE was oxidized.
The rate of aerial oxidation of the deuterated radical
oxidant probes 2, 4, 6, 8, and 10 was measured in phosphate-
buffered saline (PBS) and compared with the data for the
hydrogen analogues (see the Supporting Information). The
data in Table 1 show that the deuterated probes 2, 4, 6, 8, and
10 are significantly more stable to aerial oxidation than their
hydrogen analogues 1, 3, 5, 7, and 9, respectively. For
example, a KIE of 4.7 was observed for DDE and KIEs for
the deuterocyanines 4, 6, and 8 were in the range of 3.7–4.7.
À
These large KIE values suggest that C H bond cleavage is
involved in the rate-determining step in aerial oxidation of
these probes, and provides a methodology for improving their
stability.
Cells contain large amounts of endogenous antioxidants,
such as superoxide dismutase (SOD); radical oxidant probes
have to compete with these antioxidants in order to detect
cellular radical oxidants. A potential limitation of deuterated
radical oxidant probes is that they may react slower with
cellular radical oxidants such as superoxide radicals, and thus
will be unable to compete with cellular antioxidants. We
therefore measured the ability of the deuterated probes 2, 4,
6, 8, and 10 to compete with SOD for superoxide radicals and
compared their reactivity with their hydrogen analogues 1, 3,
5, 7, and 9 (see the Supporting Information). For these
experiments, the deuterated probes, or their hydrogen
We performed additional experiments to determine if the
greater accuracy of DDE towards the detection of radical
oxidants would improve its ability to measure radical oxidants
in cell culture. RASMs stimulated with Ang II were used for
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Table 1: KIE values (kH/kD) for aerial oxidation and oxidation by superoxide radicals for deuterated
radical oxidant probes.
Ang II or PBS as a control, and then
incubated with 10 mm of either
DDE or DHE for 10 minutes. The
cells were imaged by confocal fluo-
rescence microscopy and the mean
fluorescence intensity of the images
that contained equal numbers of
cells was quantified using Image
Pro software (see the Supporting
Information). Figure 3a–h shows
that DDE has a 2.5-fold reduction
in background fluorescence in com-
parison to DHE (3.6 vs. 9.2 fluores-
cence units), but generates only a
30% lower level of fluorescence
than DHE in Ang II stimulated
cells (6.8 vs. 9.8 fluorescence
units). Importantly, the selective
reduction of background fluores-
cence by DDE allowed the detec-
tion of radical oxidants generated
by Ang II signaling, whereas detec-
tion was not successful when DHE
was used under these conditions
(compare Figure 3d and 3h). To
further verify that DDE could be
used to detect radical oxidants, the
free radical scavenger 4-hydroxy-
2,2,6,6-tetramethylpiperidinyloxy
(TEMPOL) was added to RASMs
treated with DHE and Ang II. Fig-
ure 3g shows that TEMPOL signif-
icantly reduces the fluorescence of
RASMs treated with DHE and
Ang II, thus implying that DDE
can be used to detect radical oxi-
kH/kD
Oxidation
Probe
Product
Aerial
CÀ [b]
oxidation[a]
by (O2
)
4.7
3.7
2.5
X=H, DHE(1); X=D, DDE (2)
ethidium (11)
2.6
X=H, H-Cy3 (3); X=D, D-Cy3 (4)
X=H, H-Cy5 (5); X=D, D-Cy5 (6)
X=H, H-Cy7 (7); X=D, D-Cy7 (8)
Cy3 (12)
Cy5 (13)
Cy7 (14)
4.1
4.7
2.7
2.8
2.1
1.7
X=H, DHR (9); X=D, DDR (10)
rhodamine (15)
[a] Measured by using a 60 mm concentration of 1–10 in PBS at 258C (see the Supporting Information).
[b] kH and kD were determined following the method described by Finkelstein et al.[12] (see the Supporting
Information). Cy=cyanine dye, DHE=dihydroethidium, DDE=dideuteroethidium, DHR=dihydro- dants. In summary, the kinetic iso-
rhodamine, DDR=dideuterorhodamine.
tope effect causes DDE to generate
low background fluorescence, but
still generate similar levels of fluorescence in cells that are
stimulated to produce radical oxidants, thus resulting in
higher efficacy in comparison to DHE.
We investigated if the kinetic isotope effect would
similarly improve the ability of H-Cy7 (7) to detect radical
oxidants in vivo. H-Cy7 is a new radical oxidant probe that
can be used to detect radical oxidants in vivo because of its
high emission wavelength (765 nm); however, H-Cy7 gener-
ates moderate levels of background fluorescence in vivo,
which will potentially limit its applications. We compared the
ability of H-Cy7 and D-Cy7 (8) to image radical oxidant
Figure 2. a) Nanomolar concentrations of the hydroxyl radical (gener-
production in vivo generated by lipopolysaccharide (LPS)
stimulated acute inflammation. BALB/c mice were given
either an intraperitoneal (I.P.) injection of LPS (1 mg) or
saline for 4 hours, treated with either D-Cy7 or H-Cy7
(25 nmol) by I.P. injection, and then imaged in an IVIS
imaging system. Figure 4a–f shows that D-Cy7 is more
effective at the imaging of radical oxidants in vivo than
H-Cy7. For example, D-Cy7 had a 2.7-fold reduction in
background fluorescence in comparison to H-Cy7 (0.7 vs. 1.9
ated from Fenton’s Reaction) can be detected with DDE but not DHE.
b) Solid DDE has a greater storage life than solid DHE (see the
Supporting Information).
these experiments because of the importance of Ang II
mediated signaling in atherosclerosis.[13] RASMs were iso-
lated as previously described.[13] Cells were then cultured until
80% confluent, serum starved for 25 hours, stimulated with
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In summary, we have
demonstrated that the oxi-
dation of radical oxidant
probes by air, light, and
superoxide radicals occurs
by different mechanistic
pathways, hence leading to
different KIEs. The high
KIE values for aerial oxida-
tion of the radical oxidant
probes 1–10 suggest that
they react through a mecha-
nism that involves an exci-
plex intermediate (see the
Supporting Information). In
contrast, the low KIE values
observed for the oxidation
of 1–10 with superoxide rad-
icals suggest that this oxida-
tion occurs via either an ET–
PT–ET pathway or an
HAT–ET pathway. We
have also demonstrated
that this difference in KIEs
can be used to improve the
Figure 3. Confocal fluorescence images of RASMs treated with a) 10 mm DHE and PBS; b) 10 mm DHE and
100 nm Ang II; c) 100 nm Ang II for 4 h followed by 5 mm TEMPOL and then 10 mm DHE. d) Quantification
of fluorescence intensities from (a), (b), and (c). Confocal fluorescence images of RASMs treated with
e) 10 mm DDE and PBS; f) 10 mm DDE and 100 nm Ang II; g) 100 nm Ang II for 4 h followed by 5 mm
TEMPOL and then 10 mm DDE. h) Quantification of fluorescence intensities from (e), (f), and (g) (see the
Supporting Information). * p<0.05.
efficacy of radical oxidant probes. Deuterated radical oxidant
probes generate lower background fluorescence than their
hydrogen analogues, but generate similar levels of fluores-
cence in cells and mice stimulated to produce radical oxidants.
Based on these results, we anticipate numerous applications
of deuterated radical oxidant probes in biology and an
increased application of the KIE in biological probe develop-
ment.
Experimental Section
Determination of the kinetic isotope effects (kH/kD) for aerial
oxidation (Table 1): The procedure used to determine the kH/kD
values for the probes 1–10 is described below, using DHE and DDE
as a representative example. Stock solutions of DDE and DHE in
methanol (3.2 mm) were prepared in two separate vials, covered with
aluminum foil, and placed in a water bath maintained at 258C. At
various time points that ranged from 0–25 h, 40 mL aliquots of the
stock solutions were diluted in a 3:1 PBS/methanol solution to
generate a 60 mm concentration, and the fluorescence intensity was
measured (lex = 515 nm, lem = 559 nm). The percentage of oxidized
compound was determined by dividing the recorded fluorescence
intensity by the fluorescence intensity of a 60 mm ethidium bromide
solution. The fluorescence data were plotted as percent oxidized
versus time and the rate constants (kH and kD) were calculated
assuming first-order kinetics.
Figure 4. Fluorescence images of mice injected with a) PBS and
H-Cy7; b) LPS and H-Cy7. c) Quantification of fluorescence intensities
from (a) and (b). Fluorescence images of mice injected with d) PBS
and D-Cy7; e) LPS and D-Cy7. f) Quantification of fluorescence inten-
sities from (d) and (e).
fluorescence units, compare Figure 4d and 4a), but generated
a fluorescence level that was only 17% lower than that of H-
Cy7 in mice stimulated with LPS. As a result, D-Cy7 was
significantly better than H-Cy7 for the detection of radical
oxidants, and generated a tenfold difference in integrated
fluorescence intensity from the I.P. cavity in LPS versus
control mice, compared to only a fivefold difference for
H-Cy7 (see the Supporting Information). The kinetic isotope
effect, therefore, improves the efficacy of
Received: April 15, 2010
Revised: June 4, 2010
Published online: July 19, 2010
Keywords: fluorescent probes · imaging agents · isotope effects ·
.
D-Cy7 in comparison to H-Cy7.
oxidation · radicals
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