Kinetic analysis of nitroxide radical formation under oxygenated photolysis: Toward quantitative singlet oxygen topology

Article abstract of DOI:10.1039/c4pp00318g

Reaction kinetics for two sterically hindered secondary amines with singlet oxygen have been studied in detail. A water soluble porphyrin sensitizer, 5,10,15,20-tetrakis-(4-sulfunatophenyl)-21,23H-porphyrin (TPPS), was irradiated in oxygenated aqueous solutions containing either 2,2,6,6-tetramethylpiperidin-4-one (TMPD) or 4-[N,N,N-trimethyl-ammonium]-2,2,6,6-tetramethylpiperidinyl chloride (N-TMPCl). The resulting sensitization reaction produced singlet oxygen in high yield, ultimately leading to the formation of the corresponding nitroxide free radicals (R₂NO) which were detected using steady-state electron paramagnetic resonance (EPR) spectroscopy. Careful actinometry and EPR calibration curves, coupled with a detailed kinetic analysis, led to a simple and compact expression relating the nitroxide quantum yield Φ_R2NO (from the doubly-integrated EPR signal intensity) to the initial amine concentration [R₂NH]_i. With all other parameters held constant, a plot of Φ_R2NOvs. [R₂NH]_i gave a straight line with a slope proportional to the rate constant for nitroxide formation, k_R2NO. This establishment of a rigorous quantitative relationship between the EPR signal and the rate constant provides a mechanism for quantifying singlet oxygen production as a function of its topology in heterogeneous media. Implications for in vivo assessment of singlet oxygen topology are briefly discussed. This journal is

Full text of DOI:10.1039/c4pp00318g

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Zigler, E. C. Ding, R. Khatmullin, V. M. DiPasquale, V. F. Tarasov, R. B. Sykes and L. E. Jarocha, Photochem.
Photobiol. Sci., 2014, DOI: 10.1039/C4PP00318G.
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Kinetic Analysis of Nitroxide Radical Formation Under Oxygenated
Photolysis: Toward Quantitative Singlet Oxygen Topology
David F. Zigler, Eva Chuheng Ding, Lauren E. Jarocha, Renat R. Khatmullin, Vanessa
DiPasquale, R. Brendan Sykes, Valery F. Tarasov, Malcolm D. E. Forbes*
Department of Chemistry
University of North Carolina at Chapel Hill
Chapel Hill, NC 27599, USA
*
mdef@unc.edu
Abstract: Reaction kinetics for two sterically hindered secondary amines with singlet oxygen
have been studied in detail. A water soluble porphyrin sensitizer, 5,10,15,20ꢀtetrakisꢀ(4ꢀ
sulfunatophenyl)ꢀ21,23Hꢀporphyrin (TPPS), was irradiated in oxygenated aqueous solutions
containing either 2,2,6,6ꢀtetramethylpiperidinꢀ4ꢀone (TMPD) or 4ꢀ[N,N,Nꢀtrimethylꢀ
ammonium]ꢀ2,2,6,6ꢀtetramethylpiperidinyl chloride (NꢀTMPCl). The resulting sensitization
reaction produced singlet oxygen in high yield, ultimately leading to the formation of the
corresponding nitroxide free radicals (R NO) which were detected using steadyꢀstate electron
2
paramagnetic resonance (EPR) spectroscopy. Careful actinometry and EPR calibration curves,
coupled with a detailed kinetic analysis, led to a simple and compact expression relating the
nitroxide quantum yield ΦR2NO (from the doublyꢀintegrated EPR signal intensity) to the initial
amine concentration [R NH] . With all other parameters held constant, a plot of Φ
vs.
R2NO
2
i
[
R NH] gave a straight line with a slope proportional to the rate constant for nitroxide formation,
2 i
kR2NO. This establishment of a rigorous quantitative relationship between the EPR signal and the
rate constant provides a mechanism for quantifying singlet oxygen production as a function of its
topology in heterogeneous media. Implications for in vivo assessment of singlet oxygen
topology are briefly discussed.
1

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Introduction
Considerable interest in understanding oxidative stress mechanisms within cells has
1
driven the desire to detect reactive oxygen species (ROS) with high spatial resolution. With the
rise of the use of photosensitizers and light in the treatment of cancer (photodynamic therapy,
2
,3
PDT), significant interest has been focused on the distribution and production of molecular
1
1
1
4ꢀ7
oxygen in its lowest singlet excited states (ES) ( O = total population of ES, ꢀg + Σ ). While
2
g
several methods exist, none can directly and quantitatively measure the local population of ROS
and identify specific regions of action on the nanometer scale. The best approach to determine
singlet oxygen topology will use a diverse array of techniques to construct an image of oxidative
damage within a cell or other heterogeneous structure.
Electron paramagnetic resonance spectroscopy (EPR) can be used to detect radical
products resulting from reactions with ROS in biological media. The technique has high
8
8
sensitivity (> 10 spins per sample) and selectivity for some radical species. Observation of the
photosensitized oxidation of sterically hindered secondary amines to their EPR–active nitroxide
1
analogs is interpreted as confirmation of the presence of O (shown in Scheme 1, for example, is
2
the waterꢀsoluble photosensitizer 5,10,15,20ꢀtetrakisꢀ(4ꢀsulfunatophenyl)ꢀ21,23Hꢀporphyrin
(TPPS)).
Scheme 1
1O2
•OH
hν
2

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Recent studies of this reaction include the transformation of 2,2,6,6ꢀtetramethylpiperidinꢀ4ꢀone
(TMPD) and 4ꢀ[N,N,Nꢀtrimethylꢀammonium]ꢀ2,2,6,6ꢀtetramethylpiperidinyl chloride (Nꢀ
9
TMPCl) to their corresponding nitroxides (TEMPOne, NꢀTMPO).
Chart 1
The reaction in Scheme 1 is extensively used for qualitative confirmation of the presence
1
10ꢀ23
of O ,
however few quantitative kinetic studies have been performed. The widespread use of
2
1
this reaction for confirmation of O is surprising given the controversy over the mechanism of
2
nitroxide formation and the associated reaction kinetics. For example, several kinetic studies
have yielded contradictory rate constants (varying over four orders of magnitude) for the
2
4ꢀ32
formation of nitroxide radicals from compounds like TMPD.
Additionally, the selectivity of
1
31ꢀ37
hindered secondary amines for O has been disputed.
Clearly, the current status of the field
2
warrants a detailed examination of the nitroxide formation reaction under conditions analogous
to those used in in vitro and in vivo PDT experiments. Herewith, we report a reexamination of
the kinetics of nitroxide formation from TMPD and NꢀTMPCl under physiologically relevant
conditions. The waterꢀsoluble photosensitizer 5,10,15,20ꢀtetrakisꢀ(4ꢀsulfunatophenyl)ꢀ21,23Hꢀ
3

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porphyrin (TPPS) was used under flowing oxygen, in buffered aqueous solutions, with 395(±5)
nm illumination. After photolysis and purging with nitrogen, steady state EPR was used to
measure the concentration of nitroxide formed. Transient absorption spectroscopy (on the ns to
ms timescales) was used to understand what influence competing reactions had on the observed
kinetics.
Background
1
There are many events leading to the production and decay of O after the excitation of a
2
1
sensitizer such as TPPS, and there are several important side reactions of O with amine starting
2
1
materials or nitroxide products that should be considered in regard to O reaction kinetics. These
2
processes are summarized in Scheme 2, which is divided into additional subꢀsections for clarity.
Scheme 2
TPPS
1
2b
2
a
NP
R NH
2
kET1
1TPPS*
kNOq3
kisc1
R NH
2
kET3
3TPPS*
TPPS
TPPS
2c k
3
b
kSꢀ
3
ꢀ
3
+ O
2
k , k
kETꢀ
nrꢀ
pꢀ
1O2
3O2
R NH R NH
kR2NO
2
2
kENꢀ
3c
R NH
R NH
2
2
3
a
+
4

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Scheme 2–1: Native Relaxation
In dilute, buffered (pH = 8) aqueous solution TPPS exists in monomeric form, free from
3
8
aggregation. Excitation of TPPS occurs following absorption of light from the ultraviolet and
visible regions of the spectrum. In the absence of oxygen or other quenchers, the initially
1
populated singlet state, TPPS* (ꢀE
= 1.92 eV), relaxes to the ground state (GS) with a
(1*-GS)
lifetime τ = 10 ns. Relaxation may occur by fluorescence (k ), internal conversion (k ) or
1
f1
ic1
39ꢀ42
intersystem crossing (kisc1).
The first two processes result in repopulation of the ground state,
3
while intersystem crossing gives the lower energy excited triplet state, TPPS* (ꢀE
= 1.44
(3*-GS)
3
eV). The TPPS* state also relaxes by nonꢀradiative (k ) and radiative (k ) pathways, though
nr3
p3
3
9ꢀ41
on a much longer timescale (τ = 510 ꢁs).
3
Scheme 2–2a: Excited State Porphyrin Quenching by Amines
4
3
Hindered amine light stabilizers (HALS) like 2,2,6,6ꢀtetramethylꢀpiperidinꢀ4ꢀone, TMPD are
used to decrease damage caused by direct (type I) and singlet oxygen mediated (type II) photoꢀ
oxidation of polymers. If the amines examined in this paper react with the excited
photosensitizer, we could see inflated or deflated yields for the production of nitroxide due
formation of radical precursors or interference in singlet oxygen production. The standard
oxidation potential of TMPD has not been measured. Instead, peak potentials of an irreversible
44
+
45
oxidation are listed as 1.18 V vs. SCE and 1.14 V vs. Ag/Ag in wet acetonitrile. Electron
1
3
transfer quenching of the TPPS* and TPPS* by TMPD or similar HALS (kET1, kET3) is
thermodynamically unfavorable for amines where the first amine oxidation occurs at potentials
4
0,46,47
1
3
48
positive of <0.86 or <0.38 V vs NHE
for TPPS* or TPPS* state, respectively. Photoꢀ
oxidation of various tertiary amines or aniline by the electron deficient
5

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tetrakis(perfluorphenyl)porphyrin followed Marcus theory in dichloromethane, but no quenching
4
9
was observed of porphyrins with more electron rich phenyl substituents.
Scheme 2-2b: Energy Transfer Quenching to Nitroxide
Nitroxides like TEMPOne are efficient quenchers of excited triplet states, often with diffusion
10
−1 50.51
limited secondꢀorder rates constants (kNOq3 ~ 10 s ).
Buildꢀup of nitroxide in the solution
therefore provides an additional quenching pathway for the TPPS triplet. Due to the very low
concentrations of nitroxide produced during our photolysis experiments, it unlikely that this
pathway has much impact on our observed kinetics.
Scheme 2-2c: Energy Transfer Quenching to Oxygen (Singlet Oxygen Generation)
3
Molecular oxygen, O , efficiently quenches the triplet state of TPPS, shortening the excited state
2
5
2,53
3
−
·
lifetime to 1.9 ꢁs in air.
Single electron reduction of oxygen ( O → O , E° = −0.33 V vs.
2 2
1
3
NHE) by both TPPS* and TPPS* is thermodynamically favored and may also contribute to the
observed photobleaching of TPPS in aerated buffer (quantum yield for photobleaching Φ = 9.8
PB
−
6 54
x 10 ). Energy transfer dominates the quenching dynamics, giving the excited singlet state of
1
55
oxygen, O (k ), with high quantum yield (Φ = 0.6 in O ꢀsaturated buffer).
2
ꢀ3
ꢀ
2
Schemes 2–3a,2–3b: Singlet Oxygen Relaxation, Chemical and Physical Quenching
Singlet oxygen relaxes to the ground state rapidly by vibrational coupling to the solvent (k ) or
nrꢀ
by phosphorescence (k ) to form triplet oxygen. Due to the high frequency OꢀH stretch of water,
pꢀ
1
56
1
the lifetime of O (τ ) is 3.1 ꢁs. Ground state TPPS is nonꢀinnocent, rapidly quenching O by
2
ꢀ
2
9
−1 −1
a physical mechanism (k ). In D O, k = 2 x 10 M s and is thought to be the same in water
Sꢀ
2
Sꢀ
6

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5
5
1
−
·
(
Scheme 2–3a). There is no evidence for reduction of O to superoxide, O by ground state
2
2
1
43,57ꢀ59
1
TPPS. The reaction of O with amines is complicated.
There is evidence that O forms a
2
2
60
charge transfer complex with amines, and that this complex can decay to give either ground
state starting materials (kENꢀ, Scheme 2–3a) or some redox products (kETꢀ, Scheme 2–3b). The
1
/−
estimated 1 electron reduction potential of O (E* ≈ 0.6 V vs NHE) is likely too negative to
2
6
1
oxidize TMPD to the TMPD radical or radical cation by electron transfer.
Scheme 2–3c: Formation of Nitroxide
1
Reaction of O with TMPD produces the corresponding nitroxide (kR2NO), where kR2NO is some
2
proportion of the total amine quenching constant (k ) and may not reflect the total chemical
qꢀ
quenching pathway. This allows us to express the quantum yield for nitroxide formation (ΦR2NO
)
as eq 1:
(1)
where ΦR2NO is measured as the number of nitroxide molecules produced per absorbed photon.
Two predictions result from this kinetics expression. First, ΦR2NO should be linear with respect
1
to the initial concentration of TMPD at concentrations where the total quenching of O by
2
TMPD is much less than the sum of all other relaxation pathways. As the condition kENꢀ
[
TMPD] >> 1/τ is satisfied, the slope approaches kR2NO/ kENꢀ. If kETꢀ is truly rate limiting step,
i
ꢀ
then the apparent kR2NO = kENꢀ and the reaction will be zero order (= Φ ) at high concentrations of
ꢀ
TMPD.
Equation 1 provides a quantitative relationship between a set of known (kpꢀ, knrꢀ, kqꢀ, kSꢀ,
Φ ) and measurable ([R NH] , ΦR2NO) quantities from which a rate constant can be extracted.
ꢀ
2
i
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Below we present calibration curves showing a linear relationship between nitroxide
concentration, [TEMPOL], and integrated EPR signal intensity, quantum yield data as a function
of [R NH] and show how this information can be used, via Eq. 1, to quantitatively evaluate
2
i
kR2NO for amines of different structure. With careful consideration of all of the processes shown
in Scheme 2, coupled with the execution of high precision actinometry measurements in these
systems for the first time, our results represent the most accurate determination of this rate
constant to date. This is relevant for researchers in the field of photodynamic therapy, where
quantification and topology of singlet oxygen production are highly desirable.
Results and Discussion
The water soluble photosensitizer TPPS was chosen because it has well characterized
3
8ꢀ42
solution behavior and spectroscopic properties,
a known quantum yield of the formation of
5
3
the triplet excited state (Φ ) under our experimental conditions, and also a known quantum
T
1
55,62
yield for the formation of O (Φ ) in oxygen saturated solution.
In addition, the maximal
2
ꢀ
3
9
absorption of the TPPS Soret band overlaps well with the excitation wavelength (395 nm)
4
13 nm
5
giving highly absorbing solutions with low concentrations of photosensitizer (ε
= 3.7 x 10
−
1
−1
M cm ). The piperidine derivatives 2,2,6,6ꢀtetramethylpiperidinꢀ4ꢀone, TMPD, and 4ꢀ[N,N,Nꢀ
trimethylꢀammonium]ꢀ2,2,6,6ꢀtetramethylpiperidinyl chloride, NꢀTMPCl, were chosen because
they are water soluble and have a significant concentration of the free base at physiological pH.
3
8
A slightly basic solution (pH 8.0) was chosen to minimize TPPS aggregation and because
27
protonation of TMPD and/or NꢀTMPCl inhibits its reaction to form TEMPOne or NꢀTMPO.
The initial concentration of amines [TMPD] and [NꢀTMPCl] was calculated using the measured
i
i
pH of the photolysis solution and the HendersonꢀHasselbalch equation.
8

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Nitroxide Formation and Detection. Figure 1 shows representative EPR spectra (unnormalized)
obtained after 395 nm CW photolysis of the system in Scheme 1 at different photolysis times.
EPR signal due to nitroxide increased linearly with photolysis time. Minor background nitroxide
signal, which was present in TMPD and NꢀTMPCl samples prior to photolysis, was accounted
for by subtracting the spectrum of an identical solution kept in the dark.
0
1
min
min
2
min
5
min
5
G
Figure 1. EPR spectra collected during photolysis of 50 mM TMPD in 50 mM aqueous phosphate buffered adjusted to pH =8.0
0 minꢀblack; 1 minꢀred; 2 minꢀgreen; 5 minꢀblue). [TPPS] was kept constant at 20 mM. EPR samples were prepared by drawing
(
N deaerated solutions into 0.5 mm ID glass capillaries and sealed.
2
Omission of any of photosensitizer, amine, light or oxygen in the appropriate control
reactions gave no measureable photoproduct. EPR experiments ruled out TEMPOne production
1
3
directly from TMPD oxidation by TPPS* or TPPS*. Photolysis of TMPD and TPPS under
flowing nitrogen gave no EPR signal, nor did oxygenꢀfree photolysis followed by sparging with
O in the dark. Photolysis of TPPS under a O atmosphere and adding TMPD afterward also
2
2
1
produced no EPR–active compounds. Reactions in which known O quenchers were added
2
1
decreased the rate of production of nitroxide, further supporting a O dependent mechanism.
2
EPR Calibration Curves. To quantify the nitroxide in solution, samples were deoxygenated afer
photolysis and then observed by EPR spectroscopy. Concentration measurements were made by
comparing the integrated peak area of the nitroxide signal to a calibration curve plotting
9

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instrument response as a function of known TEMPOL nitroxide concentration (Figure 2). The
predominant source of error in the measurement of nitroxide signal was the variation in capillary
diameter (±10%), as is evident from the near constant percent deviation within a sample set.
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0
2
4
6
8
10
Concentration of TEMPOL (ꢁM)
Figure 2. A typical EPR calibration curve measured over the range of TEMPOL concentrations (0.5ꢀ10 mM) in 50 mM aqueous
sodium phosphate buffer at pH = 8.0.
Quantum Yield for Nitroxide Formation. The quantum yield of nitroxide formation (ΦR2NO) was
measured for photolysis reactions with different concentrations of TMPD and with different
concentrations of TPPS. The ΦR2NO is defined from the experimental observation of the rate of
nitroxide formation (dNR2NO/dt) relative to the rate of photons delivered (dN /dt).
A
P
proportionality constant accounts for the portion of photons absorbed as measured from the
electronic absorption spectrum of the photolysis solution. When integrated over all time for
steadyꢀstate photolysis the ΦR2NO simplifies to NR2NO/(1−I/I )·N , where N
is the number of
R2NO
o
P
photogenerated nitroxides measured by EPR and N is the total number of photons delivered
P
determined from chemical actinometry.
Although the rate of nitroxide formation varied with LED power, the measured ΦR2NO
−7
−6
was insensitive to light flux (4.3 x 10 to 4.3 x 10 einstein/s) or to photolysis time (Figures 3A
and 3B). For photolysis in which [TPPS] > 20 ꢁM, the integrated OD was > 3, giving near unit
1
0

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absorption of the delivered photons. When keeping the [TMPD] constant and correcting for
i
differences in absorbance, the ΦR2NO was independent of [TPPS] in the range of 5 to 45 ꢁM
(Figure 3C) indicating no higher order effects due to aggregation of the photosensitizer. Further,
we can assume that the k [TPPS] is much smaller than the other terms in the denominator of
Sꢀ
equation 1.
2
.5
a
2
.0
.5
.0
.5
.0
1
1
0
0
0
200
400
600
800
1000
Photolysis (sec)
2
.5
b
2
.0
.5
.0
.5
1
1
0
0
.0
0
.0
0.2
0.4
0.6
0.8
1.0
2
Avr. Irradiance (W/cm )
2
2
1
1
0
0
.5
c
.0
.5
.0
.5
.0
1
0
20
30
40
50
[
TPPS] (ꢁM)
Figure 3. Plot of TEMPOne formation quantum yield as a function of a) photolysis time; photolysis time was varied from 60ꢀ
9
4
00 sec. b) light power; Light was attenuated by adjusting the LED power supply (4ꢀ100% power) with a light flux varying from
.3 x 10 to 4.3 x 10 einstein/s; c) photosensitizer TPPS. TPPS concentration was varied from 10ꢀ45 ꢁM. The concentration of
−7
−6
TMPD·HCl was kept constant at 50 mM (37 mM free base) for a and b, and 15 mM (9.9 mM free base) in c. All solutions were
prepared in 50 mM aqueous phosphate buffered adjusted to pH =8.0. Concentration of TPPS in a and b cases was 20 ꢁM. EPR
samples were prepared by drawing N deaerated solutions into 0.5 mm ID glass capillaries and sealed.
2
Analysis of Nitroxide Formation Kinetics. Nitroxide formation proceeds from a hindered amine
in the presence of light, a photosensitizer and oxygen. Careful experiments were performed to
quantify, where possible, the important reactions outlined in Scheme 2 that may compete with
nitroxide formation. These rates or quantum yields are presented in Table 1, as are relevant
literature provided kinetic constants.
1
1

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Table 1. Selected Rate Constants, Conditions and Definitions
a
Process
Rate Constant
Conditions
Quantum yield
Ref
Singlet Photosensitizer Relaxation:
Internal conversion (k ), Fluorescence (k ), Intersystem crossing (kisc1)
ic1
f1
−1
8
−1
Singlet lifetime
τ1
1.0 x 10 s
Water
ꢀꢀ
39
Triplet Photosensitizer Relaxation:
Nonꢀradiative decay (knr3), Phosphorescence (kp3)
−1
−1
−1
3
−1
−1
−1
τ
2
.0 x 10 s
Deoxygenated
Air
ꢀꢀ
ꢀꢀ
ꢀꢀ
39ꢀ42
53
3
5
Triplet lifetime
τ3
τ3
5.3 x 10 s
6
2.2 x 10 s
O
2
t.w.
Excited Photosensitizer Quenching
Electron transfer
7
−1 −1
kET1
<10 M s
Ar or O2
Ar or O2
Air
ꢀꢀ
ꢀꢀ
t.w.
t.w.
from amine
Electron transfer
kET3
3
−1 −1
<10 M s
from amine
Energy transfer
kNOq3
7
−1 −1
6 x 10 M s
ꢀꢀ
t.w.
to nitroxide
Energy transfer to O
2
5
−1
−1
1
k
ꢀ3
5.3 x 10 s (τ
3
)
Water, O
2
0.60
56,62
(
O generation)
2
Singlet Oxygen Relaxation:
Nonꢀradiative decay (knrꢀ), Phosphorescence (kpꢀ)
−
1
5 −1
Singlet lifetime
τꢀ
2.3 x 10 s
Water
ꢀꢀ
56,60
Singlet Oxygen Quenching
Energy transfer
to porphyrin
9
−1 −1
kSꢀ
kENꢀ
kETꢀ
kNOqꢀ
2 x 10 M s
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
55
60
Energy transfer
to amine
4
−1 −1
<10 M s
CF
CF
CH
CH
OH
OH
3
3
2
2
Electron transfer
from amine
4
−1 −1
<10 M s
60
Energy transfer to
nitroxide
5
−1 −1
4.5 x 10 M s
Benzene
50,51
Nitroxide Formation
5
−1 −1
1
.6 x 10 M s
EtOH,
pH 8
ꢀꢀ
ꢀꢀ
ꢀꢀ
30
29
25
26
27
31
32
7
−1 −1
4
x 10 M s
3
−1 −1
2
.9 x 10 M s
EtOH:Benzene
pH 7
−
4
ꢀ
ꢀ
5 x 10
−
3
Nitroxide Formation
kR2NO
ꢀꢀ
pH 9
4.1 x 10
ꢀꢀ
6
−1 −1
5
.2 x 10 M s
DMSO
pH 7.4
−
7
−1
−1
−3
5
.0 x 10 M min
1 x 10
5
−1 −1
Organic
solvents
5
x 10 M s
ꢀꢀ
ꢀꢀ
37
4
−1 −1
1
.0 x 10 M s
pH 8
t.w.
a
t.w. = this work
1
2

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1
3
Transient absorption spectra show no evidence for TPPS* or TPPS* reacting directly
with the amine (Supporting Information, Figures S1ꢀS4), only rapid quenching of the triplet by
O . With and without TMPD, the transient spectra have the same features, indicating no new
2
absorbing species result from a reaction between excited TPPS and the amine. Transient decays
indicate similar dynamics with or without added TMPD (SI, Figures S5ꢀS10). In deaerated
1
buffer the lifetime of TPPS* was 11(±1) ns with and 10.1(±0.5) ns without TMPD, very close to
the literature value. The lifetimes were also similar to each other in oxygenated buffer at
11.7(±0.3) ns with and 10.7(±0.2) ns without TMPD, indicating that TMPD likely does not
1
7
quench the TPPS* state and giving an upper estimate of the quenching rate constant kET1 < 10
−
1 −1
3
M s . The kinetics of TPPS* in deaerated buffer were more difficult to interpret because the
triplet state is sensitive to even minute concentrations of oxygen, giving lifetimes of 200 to 450
ꢁ
s, but with no apparent dependence on TMPD. The lifetimes measured under flowing O were
2
much shorter, ~510 ns with or ~450 ns without TMPD. Although the fits to the decays in
oxygenated solutions appear to differ between samples with and without TMPD, the data from
each overlay one another, indicating that the variation is probably due to fitting the noisy data
and not due to added TMPD. Given all these observations, the absolute maximum of kET3 is
3
−1 −1
estimated to be less than 10 M s .
3
Quenching of TPPS* by nitroxide was also examined by TA (SI, Figure S11).
TEMPOL, a nitroxide closely related to the photoproduct TEMPOne, is a weak quencher of
3
3
TPPS*, showing enhancement of TPPS* decay with [TEMPOL] > 10 mM. At 10 mM
TEMPOL, the excited state decays at roughly 4 times the unquenched rate in aerated solution,
7
−1 −1
allowing an estimate of kqNO3 = 6 x 10 M s . Considering that the maximal concentration of
1
3

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TEMPOne achieved under the photolysis conditions in these experiments is less than 12 ꢁM,
3
TPPS* quenching by nitroxide is likely not important.
The TA experiments provide conclusive evidence that neither the starting amine nor the
nitroxide photoproduct compete with quenching of the excited photosensitizer. Therefore, it is
55,62
reasonable to use the reported Φ value, 0.60,
in Equation 1.
ꢀ
The ΦR2NO varied linearly with respect to initial TMPD and/or NꢀTMPCl concentration
(Figure 4). The linear response confirms simple firstꢀorder kinetics with respect to the amine,
1
−1
indicating that O quenching by the amine is slow relative to the normal relaxation, τ
.
2
ꢀ
4
7
3
6
2
1
5
0
0
1
2
3
4
5
6
4
3
2
1
0
[
N-TMPCl] (mM)
i
0
.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35
[
TMPD] (M)
i
Figure 4. Plot of TEMPOne formation quantum yield versus the initial concentration of TMPD. TMPD•HCl was used at
concentrations ranging from 5 to 500 mM in 50 mM aqueous phosphate buffer at pH = 8.0. The concentration of TPPS was kept
+
constant at 20 µM. The initial concentration of free base, [TMPD] , was calculated using the reported pK of TMPDH (7.6) and
i
a
the HendersonꢀHasselbalch equation. EPR samples were prepared by drawing N deꢀaerated solutions into 0.5 mm ID glass
2
capillaries and sealed. Insert: similar data plotted for NꢀTMPCl.
1
4

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The expression in equation 1 can be simplified based on the observed reaction to, Eq. 2:
(2)
where the slope of a linear fit to the data in Figure 4 is equal to the quantum yield of singlet
oxygen formation times the native lifetime in water times the rate constant for formation
5
6
55
nitroxide from freeꢀbase amine. Using τꢀ = 3.1 ꢁs and Φ = 0.60, the rate constant for
ꢀ
4
3
nitroxide formation (kR2NO) from TMPD and NꢀTMPCl is 1.0 ± 0.1 x 10 and 0.4 ± 0.1 x 10
−
1 −1
M s , respectively. The difference in rate constants between the two substrates reflects a
possible difference in driving forces. Future experiments involving deuterium kinetic isotope
effects will probe this interesting mechanistic issue further.
Conclusions
This work represents the most accurate measurements of the rate constant for nitroxide
formation for these two common substrates to date. An especially critical factor in these
determinations is the precise evaluation of the light flux hitting the samples using
standard actinometric techniques (see Materials and Methods section). In our analysis of
the kinetics (Scheme 2) we have taken into consideration the most probable competing
pathways and eliminated their involvement, leading to a simplified and compact
mathematical expression for the rate constant kR2NO. The impact of this work is its
provision of a quantitative assessment for singlet oxygen production in heterogeneous
media such as micelles, vesicles and even live cells, which can be accomplished by
running nitroxide forming reactions using isotopically labelled hindered amines with
1
5

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varying hydrophobicities. In a forthcoming paper, this topological distinction for singlet
63
oxygen production will be clearly demonstrated.
Materials and Methods
Purchased Materials. The photosensitizer 5,10,15,20ꢀtetrakisꢀ(4ꢀsulfunatophenyl)ꢀ21,23Hꢀ
porphyrin·7H O (H TPPS, TPPS) was purchased from TCIꢀAmerica (Tokyo) and the effective
2
6
formula weight (1,060 g/mol) was determined spectrophotometrically using the reported
4
0
electronic absorption spectrum. All solutions were made using MilliꢀQ water (18.1 Mꢂ/cm).
Potassium tris(oxalate)ferrate was purchased from Strem Chemical and used as purchased, or
6
4,65
was synthesized following reported procedures.
2,2,6,6ꢀTetramethylꢀ4ꢀpiperidone was
purchased from Sigma Aldrich and was recrystallized from ethanol prior to use. NꢀTMPCl was
6
6,67
synthesized according to the published procedures.
were purchased from Air Gas Company.
Oxygen and ultraꢀhigh purity argon tanks
Electron Paramagnetic Resonance Spectroscopy: EPR samples were prepared by drawing N2
deaerated solutions into 0.5 mm ID glass capillaries (Sutter Instrument) and sealed. XꢀBand
EPR spectra were recorded on a JOEL JESꢀFAꢀ100 spectrometer with 100 kHz field modulation.
Three EPR spectra were averaged per sample, with each acquired with a 15 min scan rate, 1 sec
time constant, 1.0 G modulation width, 2500 amplitude and center field of 3363.5 G.
Nitroxide Calibration: Stock solutions of (4ꢀhydroxyꢀ2,2,6,6ꢀtetramethylpiperidinꢀ1ꢀyl)oxyl
(TEMPOL) (0.5 to 10 ꢁM) were made in 50 mM sodium phosphate buffer (pH = 8.0) and
bubbled with N . The pH was adjusted using either conc. phosphoric acid or sodium hydroxide.
2
Baseline distortion for samples with a low concentration of nitroxide was corrected for by
1
6

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integrating individual hyperfine peak areas, taking great care to integrate over the same regions
for each sample.
Photochemical Nitroxide Formation and Actinometry: A typical 15 mL photolysis solution
4−
LED
contained 20 ꢁM TPPS (A
> 4), 0.5 to 500 mM TMPDꢀHCl and was buffered to pH 8.0
with 50 mM or 60 mM sodium phosphate. The initial concentration of free base, [TMPD] , was
i
+
calculated using the reported pK of HTMPD (7.6) and the HendersonꢀHasselbalch equation.
a
Photolysis were performed at ambient temperature in a 35 mL round bottom flask with vigorous
stirring to maximize oxygen exchange with pure O (g) rapidly flowing over the solution. No
2
measureable difference in the rate of nitroxide formation was observed for solutions bubbled
with O or blanketed with fast flowing O . All photolyses were performed with 395 nm (FWHM
2
2
=
10 nm) light from a light emitting diode (LED) that was transferred to the reaction vessel using
a liquid light guide, delivering approximately 2 W. Light was attenuated by adjusting the LED
power supply (4ꢀ100% power). Light flux was measured using the potassium tris(oxalate)ferrate
6
8ꢀ70
−4
chemical actinometer
and when supply power = 100%, qn,p = 2.6 x 10 einstein/min. The
4
−
concentration of TPPS was varied between 5 and 50 ꢁM, and light absorption was calculated
from the integrated product of the photolysis solution absorption spectrum (1ꢀI/I ) and the photon
o
flux normalized spectrum of the light source with respect to wavelength. Samples were
photolyzed for 1 min to 50 min and then purged with N (g) for 10 min in the dark prior EPR
2
analysis.
Acknowledgements
We thank Dr. M. Kyle Brennaman, Melissa Gish, and Prof. John M. Papanikolas for their
invaluable advice and assistance performing the transient absorption experiments. This
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7

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research made use of instrumentation funded by the UNC EFRC: Center for Solar Fuels, an
Energy Frontier Research Center supported by the U.S. Department of Energy, Office of
Science, Office of Basic Energy Sciences, under Award No. DEꢀSC00010011. MDEF
gratefully acknowledges the National Science Foundation for support of this work through
grant CHEꢀ1111873.
Electronic Supplementary Information (ESI) available: Included in the supporting
information are transient absorption spectra of solutions of TPPS with (Figures S1, S2) or
without an oxygen atmosphere (Figures S3, S4), and with (Figures S2, S4) or without
(
Figures S1, S3) added TMPD. Comparisons between transient decays for different samples
probed at a variety of wavelengths are presented (Figures S5ꢀS10) along with lifetime data
in each figure caption. Transient decays observed with added TEMPOL are presented
indicating quenching of the triplet state by the nitroxide (Figure S11). The results of
potentiometric titration of NꢀTMPCL are shown in Figure S12. Finally, synthesis of the
hindered amines and our transient absorption methods are described.
1
8

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TOC Graphic
hν
1O2
3O2
B0
2
4

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hν
1O2
3O2
B0