Phenol-based lipophilic fluorescent antioxidant indicators: A rational approach

Article abstract of DOI:10.1021/jo900335z

(Chemical Equation Presented) The reactivity, electrochemistry, and photophysics of the novel antioxidant indicator B-TOH, a BODIPY-α- tocopherol adduct, were investigated. We also studied a newly prepared BODIPY-3,5-di-tert-butyl-4-hydroxybenzoic acid ad

Full text of DOI:10.1021/jo900335z

Phenol-Based Lipophilic Fluorescent Antioxidant Indicators:
A Rational Approach
Katerina Krumova, Paul Oleynik, Pierre Karam, and Gonzalo Cosa*
Department of Chemistry, McGill UniVersity, 801 Sherbrooke Street West,
Montreal, QC H3A 2K6, Canada
gonzalo.cosa@mcgill.ca
ReceiVed July 13, 2008
The reactivity, electrochemistry, and photophysics of the novel antioxidant indicator B-TOH, a BODIPY-
R-tocopherol adduct, were investigated. We also studied a newly prepared BODIPY-3,5-di-tert-butyl-
4-hydroxybenzoic acid adduct (B-BHB) and compared the results for both sets of probes. Our results
highlight the potential of B-TOH as a fluorescent antioxidant indicator and help illustrate the considerations
to be taken into account in preparing a receptor-reporter-type fluorescent antioxidant indicator. Based
on the experimental values of the redox potentials for the reporter BODIPY and from the redox potentials
estimated for the phenol receptor segment, the off-to-on emission enhancement recently reported for
B-TOH upon peroxyl radical scavenging can be unequivocally assigned to the deactivation of an
intramolecular photoinduced electron transfer (PeT) which operates in the reduced form of B-TOH.
Theoretical calculations performed at the B3LYP/6-31G(d) level on HOMO energy levels relative to
vacuum further support the deactivation of a PeT mechanism upon peroxyl radical scavenging by B-TOH.
Fluorescence lifetimes and fluorescence quantum yields measured in a range of solvent polarities, from
hexane to acetonitrile, for B-TOH, B-BHB, and their BODIPY precursors PM605 or PMOH, are consistent
with an intramolecular nonradiative decay pathway operative in B-TOH. This pathway is not operative
in B-BHB where PeT is deemed highly endergonic based on electrochemical studies. A subsequent analysis
on the antioxidant properties of both fluorophore-phenol adducts studied herein indicates that B-TOH
antioxidant activity is on par with that of R-tocopherol, the most potent naturally occurring lipid soluble
antioxidant, whereas B-BHB is a poor antioxidant. Oxygen uptake studies upon peroxyl radical initiated
styrene autoxidation and laser flash photolysis studies on the rate of H-atom abstraction by cumyloxyl
radicals reveal similar reactivity patterns for B-TOH and 2,2,5,7,8-pentamethyl-6-hydroxychroman
(PMHC), an R-tocopherol analogue lacking the phytil tail. Analogous reactivity studies on B-BHB
underscore its poor antioxidant activity. In general, this work provides substantial amount of information
useful in designing off/on lipid soluble fluorescent antioxidant indicators based on phenol moieties.
Introduction
tion of analytes with high spatial and temporal resolution and
exquisite chemical specificity.1 Advances over the last decade
include the synthesis of receptor-reporter fluorescent probes
Chemical functionalization of organic dyes has yielded a
plethora of fluorescent chemosensors enabling real time detec-
(1) (a) Lavis, L. D.; Raines, R. T. ACS Chem. Biol. 2008, 3, 142–155. (b)
Tsien, R. Y.; Waggoner, A. In Handbook of Biological Confocal Microscopy,
3rd ed.; Pawley, J. B., Ed.; Springer: New York, 2006; pp 338-348.
* To whom correspondence should be addressed. Tel: (514) 398-6932. Fax:
(514) 398-3797.
10.1021/jo900335z CCC: $40.75
Published on Web 04/13/2009
2009 American Chemical Society
J. Org. Chem. 2009, 74, 3641–3651 3641

Krumova et al.
The choice of a chromanol receptor moiety with a structure
similar to that of R-tocopherol was based on our interest in
preparing a probe whose reaction mechanism would be on par
with that of R-tocopherol (see Scheme 1). The goal was thus to
prepare a very potent peroxyl radical scavenger. R-Tocopherol
and analogues such as trolox (a water-soluble compound where
the R-tocopherol phytil tail has been replaced by a carboxylic
acid moiety) and 2,2,5,7,8-pentamethyl-6-hydroxychroman
(PMHC, an R-tocopherol analogue lacking the phytil tail) have
been shown to undergo the series of elementary reactions shown
in eqs 1 and 2 (Scheme 1) under free-radical chain-autoxidation
conditions.4 Presumably, the same elementary reactions should
also apply to B-TOH under similar reaction conditions.
Our current objective is to gain a molecular level understand-
ing regarding the off/on switch mechanism we have observed
for B-TOH upon its reaction with free radicals in solution. We
further seek to establish the scope and limitations of B-TOH as
an off/on fluorescent antioxidant indicator, i.e., an antioxidant
which becomes emissive following free-radical scavenging by
the receptor moiety. In general, we aim at delineating the criteria
for preparing new phenol-fluorophore-based probes relying on
the off/on switch observed in B-TOH. In order to accomplish
these goals, we have performed reactivity, electrochemical, and
photophysical studies on B-TOH and on a newly synthesized
control compound, the two-segment receptor-reporter substrate
3,5-di-tert-butyl-4-hydroxybenzoic acid-BODIPY adduct (B-
BHB) (see Figure 1). These results and the ensuing discussion
are reported herein.
In the following sections, we compare the reactivity of
R-tocopherol, B-TOH, and B-BHB in the presence of peroxyl
radicals and discuss the differences in terms of the phenol O-H
bond dissociation energy (BDE). We have thus determined the
antioxidant activity of B-TOH and B-BHB by measurement of
the inhibition rate constant (during the induction periods) of
styrene autoxidation in homogeneous solution. We have also
estimated the stoichiometric factor for peroxyl radical scaveng-
ing by B-TOH and B-BHB. We have additionally conducted
competitive kinetic studies (B-TOH/R-tocopherol and B-BHB/
R-tocopherol). Finally, we have measured absolute rate constants
for H-atom abstraction from B-TOH by cumyloxyl radicals via
laser flash photolysis (LFP). These studies reveal similar
reactivity patterns for B-TOH and R-tocopherol, whereas
B-BHB is observed to lack any antioxidant properties.
FIGURE 1. Fluorophores studied.
SCHEME 1. Peroxyl Radical (ROO•) Scavenging by
r-Tocopherol (TOH)4,6 and r-Tocopheroxyl Radical (TO•)7,8
which are sensitive to reactive oxygen species (ROS).2 These
newly developed probes rely on an intramolecular photoinduced
electron transfer (PeT) off/on switch mechanism operating
between the receptor and reporter segments.3
We are working on the preparation of phenol-fluorophore
lipophilic antioxidant indicators for detecting peroxyl radicals
in the lipid membrane of live cells. Lipid peroxidation studies
both in solution but especially within live-cell membranes will
dramatically benefit from using a real time off/on fluorescent
indicator of the antioxidant status, i.e., a probe capable of
reporting via emission enhancement the depletion of peroxyl
radical scavengers and the onset of the lipid chain autoxidation.
This type of probes will ultimately allow a noninvasive spatial
and temporal monitoring of the oxidative state in live-cell
studies. We may foresee that imaging studies with specific
sensors will ultimately enable us to better understand vital links
between the chemistry and the biology of ROS.
Our results also include redox potential measurements which
together with theoretical calculations performed at the B3LYP/
6-31G(d) level on the HOMO energy levels support a photo-
induced electron transfer off/on switch mechanism. Fluorescence
lifetimes and fluorescence quantum yields measured in a range
of solvent polarities, from hexane to acetonitrile, for both
B-TOH and B-BHB and their BODIPY precursors PM605 or
PMOH are consistent with an intramolecular nonradiative decay
pathway operative in B-TOH. This pathway is not operative in
B-BHB where photoinduced electron transfer is deemed highly
endergonic based on electrochemical studies.
Having recognized that R-tocopherol (an amphiphilic chro-
manol antioxidant and most abundant form of vitamin E, see
Scheme 1) is the most active lipid soluble antioxidant present
in mammalian tissues,4 we have recently prepared the two-
segment receptor-reporter type fluorescent antioxidant indicator
B-TOH (Figure 1).5 B-TOH is an R-tocopherol analogue
provided with a lipophilic BODIPY reporter segment. Upon
scavenging peroxyl or alkoxyl radicals in homogeneous solution
B-TOH yields a highly fluorescent product.5
(2) (a) Gabe, Y.; Urano, Y.; Kikuchi, K.; Kojima, H.; Nagano, T. J. Am.
Chem. Soc. 2004, 126, 3357–3367. (b) Tanaka, K.; Miura, T.; Umezawa, N.;
Urano, Y.; Kikuchi, K.; Higuchi, T.; Nagano, T. J. Am. Chem. Soc. 2001, 123,
2530–2536. (c) Ueno, T.; Urano, Y.; Kojima, H.; Nagano, T. J. Am. Chem. Soc.
2006, 128, 10640–10641. (d) Ueno, T.; Urano, Y.; Setsukinai, K.; Takakusa,
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Chem. Soc. 2004, 126, 14079–14085.
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McCoy, C. P.; Rademacher, J. T.; Rice, T. E. Chem. ReV. 1997, 97, 1515–1566.
(4) Burton, G. W.; Doba, T.; Gabe, E.; Hughes, L.; Lee, F. L.; Prasad, L.;
Ingold, K. U. J. Am. Chem. Soc. 1985, 107, 7053–65.
The electrochemical, photophysical, and reactivity experi-
ments described herein underscore the potential of B-TOH as a
lipophilic fluorescent antioxidant indicator. Altogether, the
information provided illustrates the steps to be taken in selecting
(7) Jonsson, M.; Lind, J.; Reitberger, T.; Eriksen, T. E.; Merenyi, G. J. Phys.
Chem. 1993, 97, 8229–8233.
(5) Oleynik, P.; Ishihara, Y.; Cosa, G. J. Am. Chem. Soc. 2007, 129, 1842–
1843.
(6) Burton, G. W.; Ingold, K. U. Acc. Chem. Res. 1986, 19, 194–201.
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Chem. Res. Toxicol. 1993, 6, 351–355.
3642 J. Org. Chem. Vol. 74, No. 10, 2009

Phenol-Based Lipophilic Fluorescent Antioxidant Indicators
SCHEME 2. Homolytic Cleavage of AIBN and Styrene
Autoxidation under O2 Atmosphere15,16
thecomponentsnecessarytopreparelipophilicphenol-fluorophore-
based antioxidant indicators. The criteria are based on the redox
potentials of both receptor and reporter segments, the O-H BDE
on the receptor phenol moiety, and the photophysical and
solubility characteristics of the reporter end. This work provides
a substantial amount of information useful in the rational design
of phenol-fluorophore based off/on fluorescent antioxidant
indicators for detection of peroxyl radicals.
FIGURE 2. (A) Profiles of oxygen uptake during styrene (2.61 M)
autoxidation initiated by 19 mM AIBN in toluene at 30 °C under air.
The rate of initiation, calculated based on Arrhenius parameters for
AIBN homolysis, is Ri ) 1.98 × 10-9 M s-1. Green: Uninhibited rate
of oxidation; Ri ) 2.31 × 10-9 M s-1. Blue: Inhibition with 4.6 µM
B-BHB; τ ) 0 s, Ri ) 1.98 × 10-9 M s-1, n ) 0. Black: Inhibition
with 4.6 µM PMHC; τ ) 4670 s, Ri ) 1.97 × 10-9 M s-1, n ) 2. Red:
Results and Discussion
Inhibition with 4.6 µM B-TOH; τ ) 4500 s, Ri ) 1.98 × 10-9 M s-1
,
Two major conditions must be fulfilled by phenol-based
fluorescent antioxidant indicators, i.e., nonemissive antioxidants
which become highly emissive upon peroxyl radical scavenging.
They must have (1) a high antioxidant activity and (2) an
intramolecular off/on switch which activates following peroxyl
radical scavenging. In preparing a lipophilic phenol-fluorophore
antioxidant indicator, it is therefore of primordial importance
to evaluate the chemical reactivity of the phenol moiety toward
peroxyl radicals and to consider the mechanism by which the
resultant phenoxyl radical further decays.
Reactivity. The antioxidant activity of phenols relies on their
ability to quench peroxyl radicals via H-atom transfer from the
phenol hydroxylic group to yield a phenoxyl radical and a
hydroperoxide. The major considerations in determining anti-
oxidant activity involve the rate of hydrogen atom transfer from
phenols to peroxyl radicals and the subsequent reaction of the
phenoxyl radicals formed.4,6,7,9-12 The rates of formal abstrac-
tion of the phenol H-atom by free radicals have been shown to
be influenced by the phenol’s ring substituents as well as by
the nature of the free radical and the solvent. The reactions of
phenols with free radicals may occur by at least three different
mechanisms which have been recently reviewed.13
n ) 1.92. (B) Plot of oxygen uptake versus -ln(1 - t/τ) during the
inhibition period in the AIBN-initiated peroxidation of styrene in the
presence of (9) PMHC and (O) B-TOH. Also shown is the linear fit to
the experimental points. In the case of PMHC, the value we obtained
for kinh is 1.6 × 106 M-1 s-1. The B-TOH kinh value is 1.6-fold smaller
than that for PMHC, kinh ) 1.0 × 106 M-1 s-1 for B-TOH.
scavenging by B-TOH. As a standard, we evaluated PMHC
under identical experimental conditions (see Figure 2A,B).
The quantitative kinetic method we employed to determine
kinh has been amply discussed and applied by Barclay and
Ingold.4,9,14 Under our experimental conditions, the autoxidation
of styrene in toluene is initiated following thermolysis of AIBN15
to give two carbon-centered radicals which readily trap molec-
ular oxygen (with a reaction rate constant larger than 1 × 108
16
M-1 s-1
)
to yield two peroxyl radicals. The subsequent
propagation reactions taking place are illustrated in Scheme 2.
The differential rate equation describing the oxygen con-
sumption during the induction period is given by eq 7, where
kinh, Ri, and kp are defined by eqs 1, 3, and 6, respectively, and
where τ is the inhibition period of styrene autoxidation (τ is
determined from the intercept of the straight lines parallel to
the oxygen consumption curve during inhibition and once
inhibition is over, see Figure 2A).
Motivated by our interest in determining the antioxidant
activity for B-TOH (and the control compound B-BHB), we
measured the inhibition rate constant (or rate constant for
H-atom transfer, kinh, eq 1) of styrene autoxidation initiated by
2,2′-azobisisobutyronitrile (AIBN) in a toluene solution at 30
°C under air atmosphere (eqs 3-6 in Scheme 2). We also
determined the stoichiometric coefficient (n) for peroxyl radical
-d[O2]
dt
Ri
) kp
[H2CdCHPh] (7)
(
)
2kinh[antioxidant]
Upon integration of eq 7, we obtain eq 8 which we further
use in the data analysis. From the slope of ∆[O2]t vs -ln (1 -
t/τ) we obtain the ratio kp[H2CdCHPh]/kinh, and given the
experimental value of [H2CdCHPh] and known values of kp,
the kinh value may be readily determined.
(9) Barclay, L. R. C. Can. J. Chem. 1993, 71, 1–16.
(10) Barclay, L. R. C.; Vinqvist, M. R. Chem. Phenols 2003, 2, p 839–908.
(11) (a) Bowry, V. W.; Ingold, K. U. Acc. Chem. Res. 1999, 32, 27–34. (b)
Niki, E.; Noguchi, N. Acc. Chem. Res. 2004, 37, 45–51.
(12) Lucarini, M.; Pedulli, G. F.; Cipollone, M. J. Org. Chem. 1994, 59,
5063–70.
(13) Litwinienko, G.; Ingold, K. U. Acc. Chem. Res. 2007, 40, 222–230.
(14) (a) Barclay, L. R. C.; Ingold, K. U. J. Am. Chem. Soc. 1981, 103, 6478–
6485. (b) Barclay, L. R. C.; Locke, S. J.; MacNeil, J. M.; VanKessel, J.; Burton,
G. W.; Ingold, K. U. J. Am. Chem. Soc. 1984, 106, 2479–81. (c) Barclay, L. R. C.;
Baskin, K. A.; Dakin, K. A.; Lock, S. J.; Vinqvist, M. R. Can. J. Chem. 1990,
68, 2258–69.
(15) (a) Boozer, C. E.; Hammond, G. S.; Hamilton, C. E.; Sen, J. N. J. Am.
Chem. Soc. 1955, 77, 3233–3237. (b) Burton, G. W.; Ingold, K. U. J. Am. Chem.
Soc. 1981, 103, 6472–6477. (c) Niki, E.; Saito, T.; Kawakami, A.; Kamiya, Y.
J. Biol. Chem. 1984, 259, 4177–4182.
(16) Maillard, B.; Ingold, K. U.; Scaiano, J. C. J. Am. Chem. Soc. 1983,
105, 5095–5099.
J. Org. Chem. Vol. 74, No. 10, 2009 3643

Krumova et al.
SCHEME 3. Cumyloxyl Radical Formation and Reaction
upon ꢀ-Scission and H-Atom Transfer
t
τ
-ln 1 -
(
)
∆[O2]t ) kp
[H2CdCHPh]
(8)
kinh
Finally, the stoichiometric coefficient n is determined form
the τ value upon rearranging eq 9.
n[antioxidant]
τ )
(9)
Ri
Figure 2 illustrates the O2 consumption for styrene autoxi-
dation in the presence of B-TOH, B-BHB, and PMHC, where
the latter is known to have a stoichiometric factor n ) 2 for
peroxyl radical scavenging4 and as such serves as a standard in
our experiments. From the analysis of the data on Figure 2A,B
we obtained a stoichiometric coefficient of 1.92 for B-TOH in
comparison to PMHC. The small difference may arise in
uncertainties in determining B-TOH concentration, which was
based on a molar absorptivity at 549 nm of 70000 M-1 cm-1
for the antioxidant indicator. From eq 8 and the ratio of the
slopes in Figure 2B we determined the ratio of kinh(PMHC)/
kinh(B-TOH) to be equal to 1.6, i.e., PMHC inhibition rate
constant is only 1.6-fold larger than that of B-TOH.
In order to determine an absolute value for kinh we first need
a value for the styrene autoxidation rate constant kp in toluene.
kp is relatively insensitive to the solvent; indeed, kp for styrene
in chlorobenzene and benzene are extremely similar (kp ) 19
and 21 M-1 s-1, respectively, at 13 °C).17 Assuming kp in toluene
≈ kp in chlorobenzene ) 41 M-1 s-1 at 30 °C,17 and from the
experimental data in Figure 2B and the styrene concentration
We further estimated via laser flash photolysis (LFP) the
absolute rate constant for H-atom abstraction (kH) for B-TOH,
PMHC, and B-BHB by cumyloxyl radicals generated upon 355
nm laser excitation of cumyl peroxide (Scheme 3). Irradiation
of 1 M dicumyl peroxide in air-saturated benzene solutions with
a short (ca. 6 ns) laser pulse leads to the formation of cumyloxyl
radicals within the duration of the laser pulse (where O2
quenched any BODIPY which may have undergone intersystem
crossing upon undesired direct excitation). The cumyloxyl
radicals next react either by ꢀ-scission to give acetophenone
and methyl radical (eq 11) or by H-atom abstraction from the
phenol to form cumyl alcohol (eq 12).19 The decay rate of the
cumyloxyl radical, kexp, was indirectly monitored following the
growth rate, at 420 nm, of the phenoxyl radical derived from
PMHC or B-TOH. Phenoxyl radicals have absorption bands in
the range of 370-505 nm,20 B-TOH phenoxyl radical has an
absorption maximum at ca. 420 nm (data not shown), similar
to the absorption maximum reported for R-tocopherol phenoxyl
radical in benzene.21 As expected from the high BDE for B-BHB
and 3,5-di-tert-butyl-4-hydroxybenzoic acid, we were unable
to observe the formation of the phenoxyl radical derived from
these two phenols in the presence of cumyloxyl radical (vide
infra).
used in our experiments ([styrene] ) 2.61 M), we obtain kinh
)
1.58 × 106 M-1 s-1 for PMHC and kinh ) 1.0 × 106 M-1 s-1
for B-TOH.
The rate constant for H-atom abstraction, kH, by cumyloxyl
radical was determined from the experimental growth rate of
phenoxyl radicals under pseudo-first-order conditions at increas-
ing concentrations of the phenol substrate, according to eq 13.22
Whereas B-TOH showed an antioxidant activity similar to
that of PMHC, B-BHB did not exhibit any antioxidant activity.
These results are not surprising. The rate constant for H-atom
transfer (kinh) depends largely on the phenol O-H bond
dissociation energy (BDE).10 Unique stereoelectronic effects
give R-tocopherol a distinctively low BDE6 for the O-H bond
(78.9 kcal/mol, determined via EPR).12 The reported kinh value
for R-tocopherol in chlorobenzene is 3.2 × 106 M-1s-1.4 Similar
kinh values have been reported for PMHC (kinh ) 3.8 × 106 M-1
s-1)4 and for trolox (kinh ) 1.1 × 106 M-1 s-1)4 in the same
solvent. The smallest value recorded for trolox is consistent with
the electron-withdrawing effect of the carboxylic moiety which
strengthens the phenol O-H bond.4 A similar explanation would
account for the slightly lower reactivity we have measured for
B-TOH compared to PMHC under identical experimental
conditions. In summary, the value of kinh we have measured for
B-TOH, in the range of those listed above, is in line with a
chemical structure which preserves intact the R-tocopherol
chromanol ring leading to a low O-H BDE.
The BDE for the phenolic O-H bond in 3,5-di-tert-butyl-
4-hydroxybenzoic acid is reported to be 84.1 kcal/mol (deter-
mined via EPR).18 Such a high BDE value should in turn result
in a ca. 3-4 orders of magnitude drop in kinh at room
temperature for 3,5-di-tert-butyl-4-hydroxybenzoic acid or B-
BHB in comparison to that of R-tocopherol. As such, B-BHB
is expected to have negligible antioxidant activity in the
autoxidation of styrene, as experimentally observed.
kexp ) kꢀ + kH × [phenol]
(13)
By plotting kexp values vs increasing phenol concentration
(Figure 3), we obtained a kH value of (33 × 108) ( (1 × 108)
M-1 s-1 and a kꢀ value of (6 × 105) ( (1 × 105) s-1 for PMHC.
Similar kH values have been measured for R-tocopherol upon
reaction with tert-butoxyl radicals photogenerated in a 1:1 tert-
butyl peroxide/benzene solution.21 The high molar absorptivity
of B-TOH at 420 nm constrained the experiments to low B-TOH
concentration to ensure the probe beam could be detected in
our LFP setup; thus, only two data points were measured for
B-TOH at concentrations of 0.1 and 0.2 mM. Within the
experimental error, B-TOH is observed to be as reactive as
PMHC toward cumyloxyl radicals.
In line with the kinh values determined above for the H-atom
abstraction by peroxyl radicals, PMHC was observed to be as
reactive as B-TOH toward H-atom abstraction by alkoxyl
radicals. In the case of 3,5-di-tert-butyl-4-hydroxybenzoic acid
(19) (a) Avila, D. V.; Lusztyk, J.; Ingold, K. U. J. Am. Chem. Soc. 1992,
114, 6576–6577. (b) Avila, D. V.; Ingold, K. U.; Di Nardo, A. A.; Zerbetto, F.;
Zgierski, M. Z.; Lusztyk, J. J. Am. Chem. Soc. 1995, 117, 2711–2718.
(20) (a) Land, E. J.; Porter, G. Trans. Faraday Soc. 1963, 59, 2016–26. (b)
Das, P. K.; Encinas, M. V.; Steenken, S.; Scaiano, J. C. J. Am. Chem. Soc.
1981, 103, 4162–4166.
(21) Evans, C.; Scaiano, J. C.; Ingold, K. U. J. Am. Chem. Soc. 1992, 114,
4589–4593.
(22) (a) Cosa, G.; Scaiano, J. C. Photochem. Photobiol. 2004, 80, 159–174.
(b) Cosa, G. Pure Appl. Chem. 2004, 76, 263–275.
(17) Howard, J. A.; Ingold, K. U. Can. J. Chem. 1965, 43, 2729–36.
(18) Brigati, G.; Lucarini, M.; Mugnaini, V.; Pedulli, G. F. J. Org. Chem.
2002, 67, 4828–4832.
3644 J. Org. Chem. Vol. 74, No. 10, 2009

Phenol-Based Lipophilic Fluorescent Antioxidant Indicators
FIGURE 3. Observed growth rate constant (kexp) for the formation of
the phenoxyl radical upon 355 nm excitation of cumyl peroxide. Also
shown is the linear fit to kexp values vs [PMHC]. The growth rates were
recorded at 420 nm in the presence of increasing [PMHC] or [B-TOH]
in air-saturated benzene. The inset shows the time profiles for (O) the
absorption of the B-TOH phenoxyl radical obtained upon excitation
of cumyl peroxide in the presence of 0.2 mM B-TOH and (9) the
absorption of the PMHC phenoxyl radical obtained upon excitation of
cumyl peroxide in the presence of 0.2 mM PMHC. The larger dispersion
in the data points observed with B-TOH is due to the low intensity of
the probe beam which is significantly attenuated by absorption from
B-TOH even at the low B-TOH concentrations employed herein.
no phenoxyl radical formation was observed even at concentra-
tions of 3,5-di-tert-butyl-4-hydroxybenzoic acid ) 7.64 mM.
The kH value for 3,5-di-tert-butyl-4-hydroxybenzoic acid (and
B-BHB) is therefore at least 1 order of magnitude smaller than
that for B-TOH.
In order to establish the scope and limitations of the
prefluorescent antioxidant indicators it is important to determine
their sensing potential for peroxyl radicals. The emissive
properties of B-TOH and B-BHB were thus investigated in the
presence of peroxyl radicals generated via thermolysis of 67
mM AIBN in air-saturated toluene at 37 °C (Figure 4A-D).
Figure 4A displays the changes in fluorescence intensity
observed upon thermolysis of AIBN under increasing [B-TOH]
in toluene solutions under air. Curves are normalized to the
intensity value at time t ) 0. Concomitant with the scavenging
of peroxyl radicals by the receptor segment a linear increase in
fluorescence intensity (and a linear decrease in [B-TOH]) is
observed over time. The linear increase in intensity is consistent
with the rate law which is expected to be zero order in
antioxidant concentration. Given the measured stoichiometric
factor of ∼2 for peroxyl radical scavenging by B-TOH (vide
supra), one may show that the rate law for B-TOH consumption
follows eq 14.
FIGURE 4. Emission intensity profiles in toluene solutions incubated
at 37 °C with 67 mM AIBN for A) increasing [B-TOH]. B) [B-BHB]
) 3 µM with increasing [R-tocopherol]. C) [B-TOH] ) 3 µM with
increasing [R-tocopherol] (note that (9) consisted of 3 nM B-TOH).
All samples were air equilibrated, the fluorescence intensity was
recorded at 567 nm upon exciting at 520 nm, data points were taken
every 2.5 s, excitation and emission slits were set at 2.5 nm except for
the experiment with [B-TOH] ) 3 nM, where slits were set at 5 nm.
D) Increasing substrate concentration (either [B-TOH] for panel A or
[R-tocopherol] for panels B and C vs induction period as calculated
from the data in panels (9) A, (×) B, and (+) C.
τ directly from the intercept of the straight lines parallel to the
linear increase in intensity and the subsequent linear change in
intensity arising from BODIPY degradation. Figure 4D il-
lustrates the linear increase in τ with increasing [B-TOH]; here,
the slope is directly proportional to the stoichiometric coefficient.
One may also show that the rate of production of peroxyl
radicals upon AIBN thermolysis, Ri, may be directly estimated
from the values of τ.5
Figure 4B displays changes in fluorescence intensity observed
upon thermolysis of AIBN under [B-BHB] ) 3 µM and upon
increasing concentrations of R-tocopherol in toluene solutions
under air. Curves are normalized to the intensity value at time
t ) 0. Consistent with our expectations based on BDE values,
the receptor segment in B-BHB does not serve a protecting
antioxidant role and B-BHB undergoes peroxyl radical mediated
degradation of the BODIPY reporter segment at much the same
rate as observed for PMOH (data not shown) or for B-TOH
once the receptor segment is scavenged (see the trace obtained
with [B-TOH] ) 3 µM). Indeed, one may conclude from the
B-BHB results described above that the reaction of the peroxyl
radicals with 3,5-di-tert-butyl-4-hydroxybenzoic acid is slower
than the reaction of peroxyl radicals with the BODIPY chro-
Ri
-d[B - TOH]
)
(14)
dt
2
Upon free-radical reaction with the receptor segment, pro-
longed exposure of B-TOH to peroxyl radicals leads to a slow
decrease in fluorescence (and parallel decrease in absorption)
intensity with time (see curves obtained with low initial B-TOH
concentration in Figure 4A). This result is consistent with the
onset of radical-mediated BODIPY degradation following
the end of the induction period, i.e., upon consumption of the
antioxidant receptor segment.5 The situation is similar at high
B-TOH concentration, where, however, the drop in BODIPY
absorption concomitant with its degradation reduces inner filter
effects. Thus, rather than a drop in intensity, the degradation of
BODIPY leads to a moderate increase in intensity over time
when the initial concentration of B-TOH is larger than 15 µM.
Figure 4A underscores the tremendous potential of B-TOH
to follow in situ induction periods by simply monitoring the
emission over time. We may thus determine the inhibition period
J. Org. Chem. Vol. 74, No. 10, 2009 3645

Krumova et al.
mophores PMOH or PM605. Addition of increasing amounts
of R-tocopherol illustrate the inhibition effect in the BODIPY
degradation exerted in this case by an external (rather than
covalently linked as in B-TOH) antioxidant. The inhibition
periods τ measured for increasing [R-tocopherol] are plotted in
Figure 4D together with those recorded for B-TOH. We note
that the slopes of [substrate] vs τ are the same, within
experimental error, for B-TOH and R-tocopherol. This result is
consistent with both the prefluorescent antioxidant and R-to-
copherol having the same stoichiometric coefficient for peroxyl
radical scavenging.
antioxidant indicator. We may realize that only trace amounts
of B-TOH are necessary to follow, e.g., peroxidation of lipid
membranes in live cells containing physiological amounts of
R-tocopherol. One may alternatively conceive kinetic experi-
ments to test peroxidation in solution studies, where an in situ
simple fluorescence method relaying on B-TOH (or new
generation probes prepared under the same considerations) may
be employed.
Whereas the reaction of B-TOH with an alkoxyl radical
readily forms a phenoxyl radical with spectroscopic properties
identical to that of R-tocopherol phenoxyl radical21 (data not
shown), and whereas a similar radical is conceivably formed
following H-atom abstraction from B-TOH by peroxyl radicals,
the fate of the phenoxyl radical is more difficult to establish. In
general, the reaction of a phenoxyl radical formed upon
scavenging an initial peroxyl radical can take any of four
different pathways. They may (1) undergo rapid reaction with
the oxygen-centered radicals, (2) undergo dimerization with a
second phenoxyl radical, (3) initiate a new chain reaction upon
H-abstraction, and (4) undergo regeneration.10 Whereas path-
ways III and IV are highly improbable under high free-radical
concentration conditions, pathways I and II are certainly viable
ones. We may further rule out pathway II by considering that
this pathway is sterically demanding and should play a minor
role, if any, in the sterically crowded receptor-reporter mol-
ecules described herein.10,23
For pathway I, numerous products are expected for different
phenols, involving the addition of peroxyl radicals to the
phenoxyl radical and subsequent rearrangement. In the specific
case of the chromanoxyl radical of R-tocopherol, a chromanone
moiety which may further rearrange to a chromaquinone is the
major expected product.8,24 We have previously detected the
formation of an addition product upon B-TOH scavenging of
peroxyl radicals.5 These results were based on the UV absorption
and mass spectra of the reaction product. The nature of the
addition product (i.e., tocopherone or tocopheroquinone ana-
logue), however, remains elusive.
In closing the reactivity section, we may speculate as to the
nature of the mechanism of reaction of B-TOH with peroxyl
and alkoxyl free radicals. Three mechanisms have been recently
reviewed, which involve hydrogen atom transfer (HAT), proton-
coupled electron transfer (PCET), and sequential proton loss
electron transfer (SPLET).13 In analogy with R-tocopherol, we
expect B-TOH to follow a hydrogen atom transfer mechanism
under the experimental conditions discussed herein (low polarity
solvents). Importantly, SPLET might become the most probable
mechanism in aqueous solutions, where water makes electron
transfer far more common than in organic solvents.13 B-TOH
should certainly lend itself as a convenient substrate to perform
mechanistic studies in order to evaluate the importance of
SPLET in aqueous solution.
It is important to note herein that the fluorescence quantum
yield (Φf) for B-BHB is close to 1 (vide infra), and as such the
probe would not manifest a fluorescence enhancement even if
the receptor segment was reactive toward peroxyl radicals.
Figure 4C in turn displays changes in fluorescence intensity
observed upon thermolysis of AIBN under constant [B-TOH]
) 3 µM and with increasing concentrations of R-tocopherol in
toluene solutions under air. Rather than a linear increase in
intensity over time, as observed in Figure 4A, herein we observe
that the increase in intensity follows a sigmoid behavior. The
result is consistent with B-TOH having a slightly smaller kinh
in comparison with R-tocopherol, and therefore, the fluorescence
intensity enhancement and B-TOH consumption only occurs
once significant portions of R-tocopherol have reacted. Once
again we have determined the inhibition period τ which in this
case was given by the time at the inflection point of the sigmoid
curve (and which was extracted from the first derivative of the
fluorescence intensity vs time trajectory). The inhibition period
determined is consistent with that obtained with increasing
[B-TOH] and with that for B-BHB with increasing [R-toco-
pherol]. Close inspection of the [substrate] vs τ experimental
points obtained either with increasing [R-tocopherol] and either
constant [B-BHB] or constant [B-TOH] reveal that in order to
achieve the same τ value, the necessary [R-tocopherol] is ca. 3
µM smaller in experiments run with B-TOH vs those run with
B-BHB, consistent with B-TOH exerting an antioxidant effect
of its own.
Two additional pieces of information may be extracted from
the analysis of Figure 4C. Careful inspection of the intensity
vs time trajectories in this figure reveal that the enhancement
is significantly smaller for pure B-TOH (a ca. 8-fold fluorescence
enhancement is observed) compared to B-TOH solutions to
which R-tocopherol was previously added (a ca. 11-fold
fluorescence enhancement is observed). Whereas the intensity
vs time trajectories shown are normalized to the intensity value
at time t ) 0, in reality all trajectories reach the same final
value of fluorescence, and rather start at slightly larger
fluorescence values when [R-tocopherol] is low. The marked
difference in emission enhancement reveals that partial oxidation
of the probe occurs upon contact with the solvent and that
oxidation is prevented by sacrificial amounts of R-tocopherol
when it is first added to the solutions.
Electrochemistry. Having discussed the reactivity of B-TOH
and B-BHB and the importance of the O-H BDE in choosing
a phenol receptor segment, we discuss next the electrochemical
and photophysical properties of the probes and their precursor
reporter segments. These experiments are intended to provide
a molecular level understanding of the off/on switch mechanism
observed in B-TOH.
The second relevant result is that the fluorescent antioxidant
indicator B-TOH readily reports the onset of oxidation and the
end of the inhibition period even when in the presence of large
excesses of R-tocopherol, i.e., with a ratio of R-tocopherol/B-
TOH ∼20,000/1 (see the curve obtained with [R-tocopherol]
) 57 µM and [B-TOH] ) 3 nM). Herein the low limit for
[B-TOH] is set by the fluorimeter background, which would
make measurements at lower [B-TOH] unreliable. These results
underscore the scope and potential of B-TOH as a prefluorescent
We have previously hypothesized that an intramolecular
photoinduced electron transfer (PeT) from the receptor segment
(23) Bowry, V. W.; Ingold, K. U. J. Org. Chem. 1995, 60, 5456–67.
(24) Kamal-Eldin, A.; Appelqvist, L.-A. Lipids 1996, 31, 671–701.
3646 J. Org. Chem. Vol. 74, No. 10, 2009

Phenol-Based Lipophilic Fluorescent Antioxidant Indicators
SCHEME 4. Proposed off/on Sensing Mechanism for
B-TOH Relying on PeT from the Chromanol Moiety and
Lack of PeT from the Chromanone Moiety Generated
Following Peroxyl Radical Scavenging by the Chromanol
Moiety
FIGURE 5. Cyclic voltammograms of (A) PM605 and (B) PMOH in
degassed, Ar-saturated acetonitrile (0.1 M tetrabutylammonium hexaflu-
orophosphate) versus Fc/Fc+. Scan rate ) 200 mV s-1. The scan
direction is indicated by an arrow. The wave at a potential ) 0 V
corresponds to Fc/Fc+.
to the photoexcited reporter segment renders B-TOH nonemis-
sive. Free-radical-mediated oxidation of the chromanol receptor
segment to a chromanone or chromoquinone increases its redox
potential, thus deactivating the PeT quenching mechanism and
leading to a fluorescence enhancement (Scheme 4).5 We have
speculated that the chromanol to chromanone/chromaquinone
transformation leads to a significant increase in the redox
potential of the substrate at the receptor end in B-TOH, i.e.;
TABLE 1. Electrochemical Data for PMOH and PM605
V vs Fc/Fc+
o
-•
+•/B
EB/B
EBo
Epa1 Epa2 Epc1
Epc2 Eg (eV)
PMOH (1 V/s)
PMOH (200 mV/s) -1.56
PM605 (200 mV/s)
-1.57 0.61
2.18
NA
1.10 0.74
0.70
-1.45 -1.63 NA
EoD /D will increase upon R-tocopherone formation yielding an
There are only few electrochemical studies on BODIPY dyes;
none of these recent studies have explored the electrochemical
properties of PMOH or PM605. We therefore conducted cyclic
voltammetry experiments for both dyes in order to establish
their redox potentials.
+•
emissive molecule. Herein we have conducted experimental
work and DFT calculations to test this hypothesis.
In order to test the PeT hypothesis we calculated the standard
Gibbs free energy for the process according to eq 1525
Figure 5A shows the cyclic voltammogram for PM605
acquired in Ar-saturated 0.1 M tetrabutylammonium hexafluo-
rophosphate vs a Fc/Fc+ internal standard. In this solvent,
PM605 showed irreversible reduction and reversible oxidation
waves when scanning at 200 mV/s. Upon scanning in the
positive direction, one reversible oxidation wave was observed
for PM605 with a redox potential of +0.70 V. Upon scanning
in the negative direction, a two-electron reduction is observed
leading to two peaks with Epc values of -1.45 and -1.63 V.
Electrochemical reversibility in the oxidation was not observed
at scan rates up to 10 V/s.
Figure 5B shows the cyclic voltammogram for PMOH
obtained under identical conditions as for PM605. Contrary to
the results for PM605, PMOH showed reversible reduction and
irreversible oxidation waves at 200 mV/s. Upon scanning in
the positive direction, a two-step oxidation is observed. Elec-
trochemical reversibility in the oxidation was observed at scan
rates >1 V/s, where a redox potential of +0.61 V was
determined. Upon scanning in the negative direction, one
reversible reduction wave was observed for PMOH with a redox
potential of -1.56 V. Table 1 summarizes the electrochemical
data. The observed peak separation for the reversible waves was
∼ 60 mV demonstrating near Nernstian behavior. Also for the
reversible reduction of PMOH and the reversible oxidation of
PM605 at 200 mV/s, the peak current ratio (ipa/ipc or ipc/ipa) were
approximately unity indicating that the radical ions were fairly
stable and no additional chemical reactions took place.
The redox potentials measured herein are consistent with
those reported for the series of commercial BODIPY derivatives
∆GeoT ) [e(EDo ⁄D - EoA⁄A-•) + ω] - ∆E
(15)
+•
00
where e is the elementary charge, ω is the electrostatic work
term that accounts for the effect of Coulombic interaction of
the radical ions formed upon reduction/oxidation, ∆E00 is the
vibrational zero electronic energy of the excited fluorophore,
o
-•
EA/A is the one-electron redox potential for the electron acceptor
(BODIPY), and EoD
is the one-electron redox potential for
+•/D
the electron donor (phenol).
The Born correction term ω is usually taken as a simple
Coulombic correction given by eq 16, where q is the electron
charge, ε is the solvent dielectric constant, and R is the distance
that separate reactants at charge transfer. Values in the range
of -0.10 eV are typically used for ω in the case of acetonitrile
(ε ) 37.5). This value corresponds to a separation distance R
of ca. 0.38 nm.26
-q2
4πεoεR
ꢀ(R)
)
(16)
The vibrational zero electronic energy of the excited PM605,
∆E00, was calculated from the intersection point of the normal-
ized absorption and emission spectra for PM605 in acetonitrile,
we found a ∆E00 value of 2.22 eV (see also Table 2).
(25) (a) Rehm, D.; Weller, A. Isr. J. Chem. 1970, 8, 259–71. (b) Turro, N. J.;
Ramamurthy, V.; Scaiano, J. C. Principles of Molecular Photochemistry: An
Introduction; University Science Books: Sausalito, CA, 2009.
(26) Tachiya, M. Chem. Phys. Lett. 1994, 230, 491–4.
J. Org. Chem. Vol. 74, No. 10, 2009 3647

Krumova et al.
TABLE 2. Spectroscopic Properties for PMOH, PM605, B-TOH, and B-BHB
compd
solvent
abs λmax (nm)
Em λmax (nm)
Φf
τdec (ns)
τrad (ns)
τnr (ns)
Eg (eV)
hexanes
acetonitrile
toluene
543
536
545
557
552
560
0.832
0.838
0.995
6.57 ( 0.02
6.90 ( 0.02
6.02 ( 0.02
7.9
8.2
6.1
39.0
42.5
1187.0
2.26
2.28
2.25
PMOH
hexanes
acetonitrile
toluene
546
542
549
561
561
566
0.755
0.722
0.894
6.31 ( 0.02
6.76 ( 0.02
6.43 ( 0.02
8.4
9.4
7.2
25.8
24.3
60.8
2.25
2.26
2.23
PM605
B-TOH
B-BHB
hexanes
acetonitrile
toluene
549
544
549
555
565
567
0.063
0.039
0.137
0.44 ( 0.02
0.74 ( 0.02
0.92 ( 0.02
6.9
18.9
6.7
0.47
0.77
1.1
2.24
2.26
2.22
hexanes
acetonitrile
toluene
545
540
549
564
560
567
0.764
0.450
0.882
6.75 ( 0.02
6.79 ( 0.02
5.94 ( 0.02
8.8
15.1
6.7
28.6
12.4
50.2
2.25
2.26
2.23
+•/D
having a methyl substituent in position 8 and ethyl (PM567),
n-butyl (PM580) and isobutyl (PM597) substituents in positions
2 and 6 (note that PMOH has an hydroxyl group in position 8
window. As such, we may estimate that EDo
> 1.25 eV (vs
Fc/Fc+) for 3,5-di-tert-butyl-4-hydroxybenzoic acid, far above
the 0.76 V threshold. We may thus infer that for the system
consisting of PM605 and 3,5-di-tert-butyl-4-hydroxybenzoic
acid, intermolecular PeT will be endergonic. Consistent with
this assumption, no BODIPY quenching is observed in the
presence of 3,5-di-tert-butyl-4-hydroxybenzoic acid (vide infra).
Complementary to our experimental work, we conducted DFT
calculations. The calculations on the HOMO energy levels
relative to vacuum were performed at the B3LYP/6-31G(d)
level.30,31 Figure 8 displays the HOMO values calculated for
the reporter precursor PM605, the receptor precursors trolox
and 3,5-di-tert-butyl-4-hydroxybenzoic acid adduct (BHB in the
plot), and the oxidized receptors trolox chromanone and
duroquinone (duroquinone was chosen as a suitable model for
trolox chromaquinone). Trolox is observed to have the HOMO
at higher energy values than the HOMO of the reporter segment
(PM605), whereas oxidized trolox (either in a chromanone or
chromaquinone form) or 3,5-di-tert-butyl-4-hydroxybenzoic acid
all have their HOMO lying at significantly lower energy values
with respect to PM605. As such electron transfer to the
photoexcited receptor can only be exergonic in the case of trolox,
whereas it will be endergonic for the oxidized receptor and 3,5-
di-tert-butyl-4-hydroxybenzoic acid.
and ethyl substituents in positions 2 and 6, see Figure 1). For
o
-•
PM567, PM580, and PM597 the reported EB/B values range
between 0.53 to 0.60 V and those for EoB /B range between -1.66
+•
to -1.71 V (values originally reported vs SCE, converted herein
to values vs Fc/Fc+).27
o
-•
The one-electron redox potential for PMOH (EB/B ) was
found to be -1.56 V (vs Fc/Fc+). The EB/B value for PM605
o
-•
could not be directly determined since it undergoes an irrevers-
ible reaction. This value may however be estimated from the
spectroscopic HOMO-LUMO gap (Eg) and the electrochemical
oxidation potential value of +0.70 V obtained for PM605. In
effect, there is a good agreement between both electrochemical
(Eg ) 2.18 eV) and spectroscopic (Eg ) 2.28 eV, see Table 2)
HOMO-LUMO gaps for PMOH in acetonitrile. Assuming both
electrochemical and spectroscopic Eg values are similar for
o
PM605, we may then estimate EB/B from Eg - EBo
(see
-•
+•/B
Tables 1 and 2 for PM605 EBo and Eg values in acetonitrile,
respectively). A value of EB/B ) -1.53 V was found for PM605.
+•
/B
o
-•
Under most experimental conditions, the oxidation of phenols
occur concomitant with deprotonation of the radical cation
formed, and as such irreversible oxidation waves are generally
obtained.28 Given the difficulty in obtaining reliable one-electron
Steady-State and Time-Resolved Fluorescence. Experi-
ments performed with PM605 and increasing concentrations of
trolox or 3,5-di-tert-butyl-4-hydroxybenzoic acid are consistent
with the expectations based on eq 15. Thus, no intermolecular
quenching of PMOH is observed when 3,5-di-tert-butyl-4-
hydroxybenzoic acid is used as a quencher (see Figure 6).
Trolox, on the other hand, efficiently quenches the PMOH
emission, from the analysis of the I0/I ratio vs trolox concentra-
oxidation potential for phenols (EoD /D) we used the literature
+•
value of 0.6 V vs Fc/Fc+ recently reported by Webster et al.
for the oxidation potential of trolox ethyl ester.29
Consistent with our original hypothesis,5 PeT from trolox ester
(the electron donor D) to BODIPY (the electron acceptor A)
was found to be exergonic. We thus found that ∆GoeT is ca.
-0.15 eV for the PM605/trolox ethyl ester system in acetonitrile.
Upon rearranging eq 15, we may further estimate the
maximum value the receptor segment redox potential (EDo
)
+•
/D
(30) Gaussian 03, Revision C.02: Frisch, M. J.; Trucks, G. W.; Schlegel,
H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Montgomery, J. A., Jr.;
Vreven, T.; Kudin, K. N. ; Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi,
J.; Barone, V.; Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson,
G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa,
J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Klene, M.; Li,
X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo,
J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J. ; Cammi, R.;
Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K. ; Voth, G. A.;
Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.;
Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.;
Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.; Cioslowski,
J.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin,
R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.;
Challacombe, M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.;
Gonzalez, C.; Pople, J. A. Gaussian, Inc., Wallingford, CT, 2004.
may have which will satisfy that electron transfer to the
photoexcited reporter segment occurs spontaneously. For PM6O5
based sensors, the EDo
for the receptor segment has to be
+•/D
smaller than ca. 0.76 V in acetonitrile, i.e., EDo
∆E00 - ꢀ ) 0.76 V.
< EA/A
+
o
-•
+•/D
For 3,5-di-tert-butyl-4-hydroxybenzoic acid, the one-electron
redox potential is not observed within the acetonitrile solvent
(27) Lai, R. Y.; Bard, A. J. J. Phys. Chem. B 2003, 107, 5036–5042.
(28) (a) Moore, G. F.; Hambourger, M.; Gervaldo, M.; Poluektov, O. G.;
Rajh, T.; Gust, D.; Moore, T. A.; Moore, A. L. J. Am. Chem. Soc. 2008, 130,
10466–10467. (b) Williams, L., L.; Webster, R., D. J. Am. Chem. Soc. 2004,
126, 12441–50. (c) Webster, R. D. Acc. Chem. Res. 2007, 40, 251–257.
(29) Peng, H. M.; Webster, R. D. J. Org. Chem. 2008, 73, 2169–2175.
(31) Koch, W.; Holthausen, M. C. A Chemist’s Guide to Density Functional
Theory; Second edition ed.; Wiley-VCH: Weinheim, Germany, 2000.
3648 J. Org. Chem. Vol. 74, No. 10, 2009

Phenol-Based Lipophilic Fluorescent Antioxidant Indicators
FIGURE 8. Normalized fluorescence decay profiles for B-TOH,
B-BHB, PMOH, and PM605 in toluene. Fluorophore concentration was
ca. 3-6 µM in all cases.
FIGURE 6. HOMO energy levels (in eV) calculated at the B3LYP/
6-31G(d) level implemented through Gaussian 03.
to estimate the nonradiative decay (τnr) for the dyes according
to eq 17.
τdec
τnr )
(17)
1 - ꢁf
In the case of B-TOH, a dramatic decrease in τnr (or increase
of knr ) 1/τnr, the nonradiative decay rate constant) can be
observed (Table 2). This is consistent with the introduction of
an intramolecular quenching mechanism which is not present
in either the precursor dyes (PM605 and PMOH), or in B-BHB,
all characterized by their large τnr values. From the ratio of the
first-order rate constant knr for B-TOH intramolecular quenching
and the second-order rate constant for intermolecular quenching
kq (obtained from the quenching of PMOH by trolox) we may
further estimate, upon applying eq 18, that the effective trolox
molarity [troloxeffective] is ca. 0.5 to 1 M for B-TOH in the various
solvents studied.
FIGURE 7. Stern-Volmer plot for the fluorescence quenching of
PMOH in the presence of (9) increasing trolox and (O) increasing 3,5-
di-tert-butyl-4-hydroxybenzoic acid (BHB) in aerated acetonitrile
solution.
tion a Stern-Volmer fluorescence quenching constant KSV
)
19.3 M-1 was determined in acetonitrile (Figure 3). From the
knowledge of KSV together with the knowledge of PMOH
fluorescence lifetime in acetonitrile (τdec ) 7.2 ns), it is possible
to estimate the fluorescence quenching constant for PMOH by
trolox; kq ) 2.6 × 109 M-1 s-1.5 This quenching rate constant
is 1 order of magnitude smaller than the diffusion-controlled
rate constant kd ) 2 × 1010 s-1 in acetonitrile. The low value
of kq possibly reflects the low driving force for PeT (i.e., ∆G°eT
≈ -0.15 eV).
knr
[troloxeffective] )
(18)
kq
In summary, B-BHB, PMOH and PM605 exhibit similar
spectroscopic properties, whereas B-TOH is characterized by
significantly smaller values of φf and τdec. It follows from the
previous electrochemical, and spectroscopic studies and DFT
calculations that the B-TOH off-state arises due to an intramo-
lecular PeT from the receptor end to the reporter end.
The results discussed above illustrate what are the redox and
photophysical criteria that need to be considered in choosing a
receptor-reporter system. Based on the redox potentials char-
acterizing the receptor and reporter ends, and upon considering
the first excited singlet-state energy of the reporter, we can
estimate which receptor/reporter combinations are feasible to
undergo PeT when the receptor is in its reduced form. The
search for fluorophores readily excitable in the visible region
of the spectrum, thus avoiding autofluorescence from biological
tissue, is yet another criterion to consider in choosing the
reporter end for probes to be used in biological studies.
Whereas the emphasis on our work has been on the role of
the phenol receptor segment, it is worth noting that by replacing
the reporter BODIPY segment one may further control the extent
to which PeT is exergonic. BODIPY fluorophores where the
ethyl groups (electron-releasing substituents) at positions 2 and
The emission quantum yield (φf) of the B-BHB adduct is
similar to that of its precursor PM605, whereas the emission
quantum yield for B-TOH is more than 10-fold smaller than
that of PM605 (Table 2). These results are consistent with the
intermolecular quenching studies and the thermodynamic cal-
culations described above.
Time-resolved data acquired for B-TOH, B-BHB, PM605,
and PMOH in solutions of hexane, acetonitrile, or toluene further
illustrate that the drop in B-TOH φf arises from a dynamic
intramolecular quenching. Thus, whereas the fluorescence decay
lifetimes (τdec) of PMOH, PM605, and B-BHB are similar and
show little solvent dependence, with τdec values close to 7 ns in
all solvents (see Table 2), a significant reduction is observed
in the case of B-TOH, where τdec ) 0.44, 0.74, and 0.92 ns in
hexane, acetonitrile, and toluene, respectively (Figure 7 and
Table 2).32 From the analysis of the fluorescence decay rate
constant and the emission quantum yields it is further possible
6 are replaced by H atoms or cyano substituents lead to larger
o
-•
EA/A values. Using BODIPY fluorophores with electron-
withdrawing substituents should result in fluorescent antioxidant
indicators with improved sensitivity (i.e., larger on/off intensity
ratio). We are currently working on these probes.
(32) We note that the decay arises mostly from a short lived species, but a
secondary long lived species is also observed. We have repeated this experiment
a considerable number of times with different B-TOH batches, and in all cases
we obtain the same result, we attribute the long lived intermediate to the partial
oxidation of the probe taking place upon preparing the solution (see also the
text related to Figure 4C).
Conclusions
A comparison between the prefluorescent antioxidant indica-
tor B-TOH and the control molecule B-BHB has allowed us to
J. Org. Chem. Vol. 74, No. 10, 2009 3649

Krumova et al.
reference cell) and left to equilibrate with a water bath at 30 °C.
The volume of toluene employed was 1.3 mL in the sample cell
and 1.4 mL in the reference cell. The final styrene concentration
in the reaction chambers was 2.61 M. The initiator solution (0.1
mL of a 0.373 M AIBN solution in toluene) was injected in the
sample cell to yield a final AIBN concentration of 19 mM. A steady
rate of oxidation was next established. Known amounts of the
inhibitors in toluene (30 µL of 0.312 mM PMHC, 0.312 mM
B-TOH, and 0.321 mM B-BHB) were then injected into the sample
cell to determine their antioxidant activity. The same amount of
antioxidants was added to the reference cell so as to prevent oxygen
uptake due to self-initiation. The oxygen uptake was measured until
the rate returned to the uninhibited rate.
gain a molecular level understanding of the mechanism ac-
counting for B-TOH intramolecular emission quenching and
subsequent emission enhancement upon reaction with free
radicals. Photoinduced electron transfer is the plausible mech-
anism accounting for the BODIPY intramolecular fluorescence
quenching observed in B-TOH. Reactivity studies have under-
scored the antioxidant activity of B-TOH and the importance
of BDE in preparing a phenol based prefluorescent antioxidant
indicator. The studies reported herein allow us to delineate a
series of criteria to be considered in selecting the receptor and
reporter components to prepare phenol-based lipophilic fluo-
rescent antioxidant sensors.
Laser Flash Photolysis Studies. Experiments were carried out
in a commercially available Luzchem 212 LFP setup provided with
a Tektronix TDS 2000 digitizer for signal capture. A Nd:Yag laser
(Continuum model Surelite I-10) was used for excitation at a
wavelength of 355 nm. The laser was attenuated yielding 9 mJ
laser pulses with a pulse width of ∼6 ns. Cuvettes, 1 cm in path
length, were utilized in all experiments, the solutions consisted of
1 M dicumylperoxide in benzene to ensure an absorption of 0.3 at
the excitation wavelength. We chose 355 nm as the excitation
wavelength to minimize direct excitation of B-TOH (molar ab-
sorptivity ε355nm ) 3.4 × 103 for B-TOH). Air saturated solutions
were utilized to rapidly quench any triplet BODIPY which may
form under our experimental conditions, experiments were con-
ducted at room temperature. The growth of the phenoxyl radical
was monitored at 420 nm following laser excitation of the
dicumylperoxide solutions containing increasing concentrations of
PMHC or B-TOH. In order to minimize sample degradation, fresh
samples were irradiated with a total of 10 laser shots to acquire
the ∆OD temporal evolution and the kexp for the various antioxidant
concentrations, the solutions were further agitated in-between shots
(prolonged irradiation of the solutions containing B-TOH and
dicumyl peroxide (12 or more laser shots) lead to an observable
emission enhancement arising from B-TOH oxidation upon reaction
with cumyloxyl radicals).
Steady-State Fluorescence Studies. A Cary Eclipse Spectro-
photometer with temperature controller was utilized to measure the
emission intensity profiles of 3 µM B-TOH and 3 µM B-BHB
solutions in toluene. Fluorescence was recorded at 567 nm upon
exciting at 520 nm using 2.5 nm excitation and emission slits.
Toluene solution (2.2 mL) containing B-TOH or B-BHB with or
without R-tocopherol were incubated for 10 min at 37 °C before
804 µL of AIBN 250 mM in toluene were added to each cuvette
for final AIBN concentration of 67 mM. The emission intensity
was followed at 2.5 s intervals for 8000 s.
The choice has to be primarily based on the redox properties
of the phenol and fluorophore segments as well as the first
excited singlet state energy of the latter in order that PeT from
the phenol to the fluorophore is exergonic. Intermolecular
quenching via PeT should ideally be diffusion controlled; this
will ensure that upon tethering the reporter and receptor segment
a significant decrease in the emission quantum yield of the
former will be observed given the high effective quencher
concentrations achieved following covalent binding. Our interest
in preparing lipophilic fluorescent antioxidants to monitor the
oxidative stress in lipid bilayers reduces the choice of fluoro-
phores mostly to those derived from BODIPY. Cyanine dyes
among others, frequently bearing a formal charge, are not
suitable candidates for lipophilic fluorescent antioxidant indica-
tors, however they would certainly be suitable candidates to be
considered in preparing water-soluble antioxidant indicators.
The chemical reactivity of the phenol moiety plays a
significant role in the choice of the receptor end. Whereas
phenols containing a low BDE hydroxyl group readily scavenge
peroxyl radicals, phenols with a high BDE hydroxyl group are
poor antioxidants. Interestingly, the BDE in phenols decreases
upon substituting H-atoms with electron donating groups in the
aromatic ring and it increases upon substituting H-atoms with
electron-withdrawing groups.18 The former substitution pattern
further favors PeT from the phenol to the fluorophore moiety,
whereas the later disfavors this PeT mechanism as can be
observed with B-TOH and B-BHB.
The peroxyl radical mediated degradation observed for
PMOH or B-BHB, albeit slow, further underscores the necessity
of choosing a fluorophore whose chemical structure minimizes
the possibility of side reactions with free radicals.
The emission quantum yield for PM605 in ethanol (φs ) 0.74)
was used as the standard to calculate the emission quantum yield for
the various dyes in the different solvents (φu). Optically matched
solutions of PM605 in ethanol and of a dye of interest were excited
at the same wavelength, and the emission recorded and integrated
over all the wavelength range. The emission quantum yield was
calculated according to eq 19, where A stands for the absorption,
I for the integrated intensity, and n for the refractive index for the
unknown (u) or the standard sample (s).
Experimental Section
Materials. 8-Acetoxymethyl-2,6-diethyl-1,3,5,7-tetramethylpyr-
romethene fluoroborate (PM605) was purchased from Exciton, Inc.
(()-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid
(trolox), 3,5-di-tert-butyl-4-hydroxybenzoic acid, N-(3-dimethy-
laminopropyl)-N′-ethylcarbodiimide (EDC), dimethylaminopyridine
(DMAP), 2,2,5,7,8-pentamethyl-6-hydroxychroman (PMHC), di-
cumyl peroxide, 2,2′-azobis-(2-methylpropionitrile) (AIBN), and
R-tocopherol were used as received; styrene was distilled prior to
use. Solvents were used without further purification unless otherwise
specified in the text.
AsIun2u
AuIsn2s
ꢁu ) ꢁs
(19)
Electrochemical Studies. Voltammetric experiments were con-
ducted with a computer-controlled CHI760C potentiostat with a
BASi C3 cell stand. The working electrode was a 2 mm Pt electrode
with a Pt wire auxiliary electrode and a 0.01 M Ag/AgNO3 solution
reference electrode. A 0.1 M solution of tetrabutylammonium
Absorption. Absorption spectra were recorded employing 1 cm
× 1 cm quartz cuvettes.
Oxygen Uptake Studies. Autoxidation studies were carried out
at 30 °C under air (760 Torr) in a dual-channel oxygen uptake
apparatus equipped with a sensitive pressure transducer described
previously.33
(33) Wayner, D. D. M.; Burton, G. W. In Handbook of Free Radicals and
Antioxidants in Biomedicine; CRC Press: Boca Raton, 1989; Vol. 3, pp 223-
232.
Known volumes of substrate (styrene) and solvent (toluene) were
placed in both reaction chambers of the system (sample cell and
3650 J. Org. Chem. Vol. 74, No. 10, 2009

Phenol-Based Lipophilic Fluorescent Antioxidant Indicators
hexafluorophosphate in dry acetonitrile was used as the electrolyte
solvent, in to which the species of interest were dissolved for
analysis. The solution was purged with argon with simultaneous
stirring and left under a blanket of argon. All values are reported
vs ferrocene, with the oxidation of ferrocene explicitly measured
and corrected to zero for each experiment.
Fluorescence Lifetime Studies. The fluorescence lifetime
measurements were carried out using a Picoquant Fluotime 200
time correlated single photon counting setup employing an LDH
470 ps diode laser (Picoquant) with excitation wavelength at 470
nm as the excitation source. The laser was controlled by a PDL
800 B picosecond laser driver from Picoquant. The excitation rate
was 10 MHz, and the detection frequency was less than 100 kHz.
Photons were collected at the magic angle.
Computational Methods. All structures were computed using
the density functional theory at the B3LYP/6-31G(d) level imple-
mented through Gaussian 03.30 Several starting geometries were
utilized to get geometry optimization in order to ensure that the
optimized structure corresponds to a global minimum.
Synthesis. 8-Hydroxymethyl-2,6-diethyl-1,3,5,7-tetramethylpyr-
romethene fluoroborate (PMOH) and 8-((() 6-hydroxy-2,5,7,8-
tetramethylchromane-2-carbonyloxy)methyl-2,6-diethyl-1,3,5,7-
tetramethylpyrromethene fluoroborate (B-TOH) were prepared as
described in the literature.5,34
3,5-Di-tert-butyl-4-hydroxybenzyloxymethyl-2,6-diethyl-1,3,5,7-
tetramethylpyrromethene Fluoroborate (B-BHB). To 5 mL of
anhydrous CH2Cl2 under argon atmosphere and constant stirring
were added PMOH (15.5 mg, 0.0464 mmol, 1 equiv), 3,5-di-tert-
butyl-4-hydroxybenzoic acid (11.6 mg, 0.0463 mmol, 1 equiv), EDC
(8.9 mg, 0.046 mmol, 1 equiv), and DMAP (5.1 mg, 0.042 mmol,
0.9 equiv). The resulting solution was refluxed under argon in the
dark for 1 h. The reaction mixture was then cooled to room
temperature and quenched with 10 mL of saturated aqueous solution
of NH4Cl. The phases were separated, and the aqueous phase was
extracted with CH2Cl2 (10 mL × 3). The combined organic fractions
were dried with anhydrous MgSO4, followed by concentration under
reduced pressure. Purification by flash chromatography on silica
gel was performed as follows: The dispersion of the crude product
in hexanes was prepared and loaded on the column. It was first
flushed with hexanes and then eluted with a 1/1 CH2Cl2/hexanes
mixture, dried under vacuum, dissolved in ethyl acetate, and washed
10 times with 10% K2CO3 (aq) to afford a coupling product as a
1
dark purple solid in 45% yield: H NMR (400 MHz, CDCl3) δ
ppm 8.05 (s, 4H), 5.79 (s, 1H), 5.55 (s, 2H), 2.54 (s, 6H), 2.41 (q,
J ) 7.6 Hz, 4H), 2.33 (s, 6H), 1.50 (s, 18H), 1.35 (s, 18H), 1.06
(t, J ) 7.6 Hz, 6H); 13C (75 MHz, CDCl3) δ ppm 166.9, 158.8,
154.9, 137.0, 136.2, 133.7, 132.8, 132.4, 127.5, 120.3, 58.8, 34.7,
30.5, 17.5, 15.1, 13.1, 13.0; LRMS (EI, 70 eV) m/z 566.4 (M+
100), 303.2 (30), 235.1 (54); HRMS (EI) for C33H45N2O3BF2 (M+)
calcd 566.34913, found 566.35017; IR (neat) ν cm-1 3586 (m),
2963 (m), 2934 (m), 2876 (m), 1713 (m), 1556 (s), 979 (s);
absorption λmax ) 549 nm in toluene; emission λmax ) 567 nm in
toluene.
Acknowledgment. We are grateful to McGill University, the
Natural Sciences and Engineering Research Council of Canada,
the Canada Foundation for Innovation New Opportunities Fund,
the Fonds Que´be´cois de la Recherche sur la Nature et les
Technologies-Nouveaux Chercheur Program and the Centre for
Self-Assembled Chemical Structures for financial assistance.
K.K. and P.K. are also thankful to the McGill Chemical Biology
Fellowship Program (CIHR) for postgraduate scholarships. We
thank Mr. Yoshihiro Ishihara for assistance with the preparation
of B-BHB. We also thank Prof. D. Perepichka at McGill
University for help with the electrochemical studies and DFT
calculations. We are also thankful to Prof. J. C. Scaiano and
Ms. V. Filippenko at the University of Ottawa for assistance
with the oxygen uptake measurements.
Supporting Information Available: 1H NMR and 13C NMR
spectra for B-BHB. This material is available free of charge
via the Internet at http://pubs.acs.org.
(34) Amat-Guerri, F.; Liras, M.; Carrascoso, M. L.; Sastre, R. Photochem.
Photobiol. 2003, 77, 577–584.
JO900335Z
J. Org. Chem. Vol. 74, No. 10, 2009 3651