The excited-state interaction of resazurin and resorufin with amines in aqueous solutions. Photophysics and photochemical reactions.

Article abstract of DOI:10.1562/0031-8655(2002)0760385TESIOR2.0.CO2

The photophysics and photochemical behavior of the phenoxazin-3-one dyes, resazurin and resorufin, have been studied in aqueous solutions. The irradiation of resazurin in the presence of amines leads to deoxygenation of the N-oxide group, giving resorufin. This photoreaction is highly dependent on the amine structure and is efficient only in the presence of tertiary aliphatic amines. The absorption and fluorescence properties of these dyes are dependent on pH. At pH above 7.5 both dyes are in their anionic form. For resorufin this form is highly fluorescent (phiF = 0.75). At lower pH the fluorescence is strongly reduced. The N-oxide dye presents a very weak fluorescence quantum yield (0.11), which also is reduced at low pH. Flash photolysis experiments allowed characterization of the triplet state and the transients formed after irradiation of these dyes in the absence and presence of amines. The triplet quantum yields are 0.08 for resazurin and 0.04 for resorufin. The photodeoxygenation of N-oxide in the presence of amines occurs from the triplet state.

Full text of DOI:10.1562/0031-8655(2002)0760385TESIOR2.0.CO2

Photochemistry and Photobiology, 2002, 76(4): 385–390
The Excited-State Interaction of Resazurin and Resorufin with Amines
in Aqueous Solutions. Photophysics and Photochemical Reaction
{
1
C. Bueno , M. L. Villegas , S. G. Bertolotti , C. M. Previtali , M. G. Neumann and M. V. Encinas*
1
2
2
3
1
1
Facultad de Qu ´ı mica y Biolog ´ı a, Universidad de Santiago de Chile, Santiago, Chile;
Departamento de Qu ´ı mica F ´ı sica, Universidad Nacional de R ´ı o Cuarto, Argentina and
Instituto de Qu ´ı mica de Sao Carlos, Universidade de Sao Paulo, Sao Carlos, SP, Brazil
2
3
Received 22 April 2002; accepted 9 July 2002
ABSTRACT
The photophysics and photochemical behavior of the
phenoxazin-3-one dyes, resazurin and resorufin, have been
studied in aqueous solutions. The irradiation of resazurin in
the presence of amines leads to deoxygenation of the N-oxide
group, giving resorufin. This photoreaction is highly de-
pendent on the amine structure and is efficient only in the
presence of tertiary aliphatic amines. The absorption and
fluorescence properties of these dyes are dependent on pH.
At pH above 7.5 both dyes are in their anionic form. For re-
F
sorufin this form is highly fluorescent (F 5 0.75). At lower
pH the fluorescence is strongly reduced. The N-oxide dye
presents a very weak fluorescence quantum yield (0.11),
which also is reduced at low pH. Flash photolysis experi-
ments allowed characterization of the triplet state and the
transients formed after irradiation of these dyes in the ab-
sence and presence of amines. The triplet quantum yields
are 0.08 for resazurin and 0.04 for resorufin. The photodeoxy-
genation of N-oxide in the presence of amines occurs from the
triplet state.
anol. It was found that a clean photodeoxygenation to resorufin
occurs from the quenching of the triplet state of the dye (9).
The photoinduced deoxygenation of N-oxides has been
reported previously for other molecules (10) and was found to
proceed on irradiation of N-oxide alone or in the presence of
suitable acceptors such as amines (11,12). But in most cases the
reaction is accompanied by side reactions and rearrangements of
the N-oxide (13). More recently, we found that the two related
dyes, resazurin and resorufin, in the presence of triethanolamine
(TEOHA) produce radicals that are efficient photoinitiators of
aqueous acrylamide polymerization (14).
Because photophysical data for these phenoxazin-3-one dyes are
scarce in the literature, and the reactions of the excited state with
amines have been even less studied, we undertook a detailed study
of the properties of the excited states of resazurin and resorufin in
aqueous solution, as well as their photoreactions with amines.
INTRODUCTION
Resazurin is a phenoxazin-3-one dye widely used for testing var-
ious biological materials (1,2). In many of these uses resazurin
EXPERIMENTAL PROCEDURES
(
I) is reduced to resorufin (II), changing its color from blue to
Chemicals. Resazurin and resorufin were obtained from Aldrich (Mil-
waukee, WI) and were used as supplied. The amines (Aldrich) were puri-
fied by vacuum distillation just before their use. Water was purified
through a Millipore Milli-Q system.
Measurements. Dye solutions were purged with argon for 30 min
before their use. The pH was adjusted by addition of NaOH or HCl once
all the other reagents were added. The photoreduction of dyes by amines
was studied spectrophotometrically in aqueous solution at pH 9.5. Resa-
zurin samples were irradiated at 615 nm and resorufin samples at 572 nm
with a Photon Technology International irradiation system (Lawrence-
ville, NJ) comprising a 150 W xenon lamp and a monochromator (PTI-
pink (1,3). The photochemical deoxygenation of heterocyclic
N-oxides also has been considered as a mechanistic model for
oxygen atom transfer in enzymatic processes involving mono-
oxygenases (4–6). In contrast, the photophysics of these dyes has
been much less explored. Resorufin has been used as a probe
molecule to study the reorientation of solvent molecules (7,8). In
a previous communication we reported on the photophysics and
photochemical reaction of resazurin with tripropylamine in meth-
1
02). The rate of the photoreaction was measured after the decrease of
the absorbance of dyes at different irradiation times. The photoreaction
quantum yield was determined taking Aberchrome 540 as a reference
(15). Absorption spectra were determined on a Hewlett–Packard 6453E
diode array spectrophotometer.
Steady-state fluorescence measurements were made using a Fluorolog-
Spex spectrofluorimeter. Fluorescence quantum yields were determined
relative to cresyl violet in methanol (16). Fluorescence lifetime measure-
ments were performed with an Edinburgh Instruments OB 900 time-
correlated single-photon counting fluorometer. The singlet quenching rate
{
Posted on the web site on 4 September 2002.
*
To whom correspondence should be addressed at: Facultad de Quimica
y Biologia, Universidad de Santiago de Chile, Casilla 40, Correo 33,
Santiago, Chile. Fax: 562-681-2108; e-mail: mencinas@lauca.usach.cl
Abbreviations: Am, amine; DABCO, 1,4-diazabicyclo[2.2.2]octane; TEO-
HA, triethanolamine; ZnTPP, 5,10,15,20-tetraphenyl-21H,23H-porphine
Zn.
Ó 2002 American Society for Photobiology 0031-8655/02
$5.0010.00
3
85

3
86 C. Bueno et al.
Table 1. Photophysical parameters of the dyes in water at pH 9.5
Resazurin
Resorufin
2
1
21
Absorption, kmax (e [M cm ])
Fluorescence, kmax
602 nm (47 000)
634 nm
0.11
572 nm (56 000)
585 nm
0.74
U
F
s
U
F
0.7 ns
0.08
2.9 ns
0.04
700 nm (10 000)
T
2
1
21
Triplet, kmax (e [M cm ])
350 nm (4100);
8
25 (4500)
When excited at 602 nm, resazurin presents a fluorescence
spectrum with an unstructured and broad band with a maximum
at 634 nm. The fluorescence intensity presents strong pH depen-
dence, but the spectral shape remains unaltered. The emission re-
mains almost unchanged from pH 7.5 to 12, and a fluorescence
quantum yield of 0.11 was determined. Further decrease in pH
strongly reduces the fluorescence intensity, so that almost no
fluorescence was detected for resazurin in high acid solution
even when excited at 520 nm. From a plot of fluorescence quan-
tum yield against pH we estimated a pK value of 5.5. This value
is close to that obtained from the absorption titration, indicating
that the change of fluorescence with pH reflects the change of
the ground state.
Figure 1. Absorption spectra of resazurin in water at different pH.
constants were measured by following the decrease of the fluorescence
intensity or the emission decay elicited by the amine addition.
Transient absorption measurements were made using laser flash photol-
ysis equipment. The harmonic wavelengths of a Nd:YAG laser (525 or
3
55 nm, 5 mJ/pulse, 20 ns) were used for sample excitation. The signals
from the monochromator–photomultiplier system were initially captured
by a HP54504 digitizing oscilloscope and then transferred to a computer
for storage and analysis. Measurements were performed in samples sub-
jected to continuous argon bubbling.
Resorufin excited at 570 nm exhibited a strong fluorescence
emission with a broad band centered at 585 nm. This band re-
mains almost unchanged between pH 12 and 7.5, with fluores-
cence quantum yield of 0.74. A progressive decrease in intensity
without change in the band shape was observed from pH 7 to 4.
At lower pH almost no fluorescence was detected. As in the case
of resazurin, the pK value for resorufin obtained from the fluo-
rescence titration curves is very close to that obtained from the
UV-absorption measurements. This suggests that during the life-
time of the singlet excited state of these dyes, no different proto-
tropic equilibrium is established with respect to that already
present in the ground state.
RESULTS AND DISCUSSION
The UV-visible absorption spectrum of resazurin in basic aque-
ous solutions consists of an intense absorption band at 602 nm
2
1
21
(
e ¼ 47 000 M cm ) and a weak band at 380 nm. This spec-
trum is similar to that reported in methanolic solution (9). The
bands are assigned to the pp* transition of the phenoxazin-3-one
and to the weak np* transitions of the N-oxide, respectively.
Figure 1 shows the pH dependence of the visible absorption
of resazurin. The spectrum is almost insensitive to pH changes in
the range of pH 7.5–11. At 602 nm the absorption intensity in-
creases linearly with the concentration (0.5–20 lM). This indi-
cates that the aggregation of the dye is not appreciable in this
concentration range. At pH between 7 and 3.5, the absorption
sharply decreases, and a new broad absorption band appears in
the 400–600 nm region, with an isosbestic point at 534 nm.
From a plot of absorption at 602 nm against pH, a pK value of
The fluorescence lifetimes in water at pH 9.5, evaluated from
the emission time profiles, were 0.7 and 2.9 ns for resazurin and
resorufin, respectively. The rather long lifetime of resorufin, as
compared with that of resazurin, is in agreement with the high
fluorescence quantum yield. These values give inherent radia-
8
21
tive rate constants for the emitting state of 1.2 3 10 s and
8
21
2
.5 3 10
s
for resazurin and resorufin, respectively, which
are consistent with a highly allowed pp* electronic transition.
The photophysical data for both dyes are summarized in Table 1.
The photophysical parameters for resorufin are comparable in
magnitude with those reported by Flamigni et al. (8) (s ¼ 5.5
5
Further changes in the spectrum are observed at pH lower than
.5 was obtained, which is assigned to the oxygen protonation.
2
.0, indicating the protonation of the N-oxide group.
The visible spectrum of resorufin is characterized by an
2
1
21
and 4.9 ns in nitrile and alcoholic solvents, respectively; U
F
¼
intense band centered at 572 nm (e ¼ 56 000 M cm ) with
a shoulder at 535 nm. As in the case of resazurin the absorbance
and shape of the spectrum are unaffected in the pH 12–7.5 inter-
val. At lower pH the absorption decreases drastically, and a new
band with a maximum at 485 nm appears. From a plot of the ab-
sorbance at 572 nm against pH, a pK value of 5.8 was obtained.
This value is very close to that obtained for resazurin and is at-
tributed to the oxygen protonation. Similar spectral changes have
been reported for resorufin by Flamigni et al. (8). They attributed
the alteration of the spectrum at pH ,5 to the aggregation of the
protonated species. But we found that the absorbance of the band
at 485 nm follows the Lambert–Beer law, which indicates insig-
nificant aggregation under our experimental conditions.
0
.63 in alcohol).
Transient absorption spectra
When an argon-saturated aqueous solution of rezasurin at pH 9.5
is laser flash irradiated at 532 nm, an absorption transient spec-
trum is obtained. Figure 2 shows the time-resolved differential
absorption spectra at 1 and 90 ls after the laser pulse. At 1 ls,
two bands can be seen, one in the 650–800 nm region and the
other at 300–490 nm. At longer lifetimes the absorption in the
650–850 nm region and at 350 nm are greatly reduced, although
an absorption remains at 400 nm, and a new band can be

Photochemistry and Photobiology, 2002, 76(4) 387
Figure 3. Transient absorption spectrum of resorufin in water at pH 9.5
recorded 4 ls (d) and 75 ls (s) after the laser pulse. Insert: kinetic pro-
files at 700 and 400 nm.
Figure 2. Transient absorption spectrum of resazurin in water at pH 9.5
recorded 1 ls (d) and 90 ls (s) after the laser pulse. Insert: kinetic pro-
files at (a) 400 nm; (b) 825 nm; and (c) 480 nm.
dominantly to the thioxanthone. The transient absorption spec-
trum gave the same band in the region of 600–800 nm as that
obtained by direct excitation of the dye at 532 nm.
The sensitization was also carried out using benzophenone. The
triplet of benzophenone decays (525 nm) is shortened in the
presence of resorufin, with a bimolecular rate constant of 5 3
observed at ;480 nm (Fig. 2). The negative absorption (500–
620 nm) and the valley at 379 nm indicate the depletion of the
ground state. The absorption in these regions shows a different
intensity and profile decay. The inset in Fig. 2 shows that the
signal at 825 nm decays with a clear first-order kinetics with life-
time of several 10s of microseconds. This trace is superimposed
on that measured at 350 nm. The decay of the signal at 400 nm
is similar to that at 825 nm, but a long-lived absorption remains.
On the other hand, the signal at 480 nm increases with time at
a rate similar to that of the decay of the species at 825 nm
9
21 21
1
constant in water.
0 M
s
. This value is close to the diffusion-controlled rate
The decays at 450 and 380 nm follow a decay similar to that
at 700 nm. On the basis of the decay kinetics and the sensitiza-
tion experiments, the spectrum at 4 ls in Fig. 3 is assigned to
the combined T–T absorption and the ground-state bleaching of
resorufin. The first-order rate constant measured at 700 nm was
also dependent on the ground-state dye concentration. The self-
quenching rate constant, evaluated from the linear plots of kobs
(Fig. 2). This implies that the absorption at 480 nm is caused by a
species formed by the reaction of the species absorbing at 825 nm.
The spectrum recorded at 1 ls can be ascribed to the triplet
state of the dye. This is based on the fact that O and other trip-
2
let quenchers such as dimethylaniline effectively quench the
bands at 825 and 350 nm. Furthermore, a similar absorption
spectrum in the 600–800 nm region was found when benzophe-
none was excited in the presence of resazurin. The decay of the
band at 825 nm depends on the ground-state concentration. The
bimolecular rate constant for the quenching of resazurin triplet
by the ground-state dye was determined from the decay rate con-
stant at various concentrations of the dye by using low laser
9
21 21
vs the resorufin concentration, was 1.2 3 10
M
s . This
value is two times higher than that obtained for the N-oxide dye.
As in the case of resazurin, the spectrum at 75 ls in Fig. 3 is
ascribed to a small residual absorption of the triplet state and to
the semireduced and the semioxidized forms of the resorufin in
the 350–480 nm region.
The molar absorption coefficients of triplet dyes were deter-
mined by the total depletion method. In both cases the negative
absorption of the difference transient spectra matched the
ground-state band. This is consistent with the lack of photoprod-
uct formation under our conditions of laser experiments. Consid-
ering that in the region of ground-state absorption the molar
absorption coefficient of the triplet state is negligible, and using
the measured absorption coefficient for the dye ground-state ab-
8
energies. The rate constant for the self-quenching was 4.9 3 10
2
1 21
M
tions from the ground-state dye according to Eq. 1
s
. The self-quenching may involve electron transfer reac-
3
ꢀ
ꢁ
ꢀ
ꢂ 2ꢀ
þ Dꢂ
ðD Þ þ D ! D
ð1Þ
ꢂ22
ꢂ
where D
and D represent the semireduced and semioxidized
2
1
21
forms of the dye. Accordingly, the spectrum at 90 ls with appar-
ent maxima at 400 and 480 nm can be assigned to the combined
absorption of these species.
sorption, e of 4500 6 1000 M cm (825 nm) and 10 000 6
T
1
2
21
2000 M cm (700 nm) were obtained for the T–T absorption
of resazurin and resorufin, respectively.
Laser excitation at 532 nm of resorufin aqueous solution at pH
.5 results in the absorption spectra presented in Fig. 3. At short
The molar absorption coefficient of resorufin was also evaluat-
ed from the sensitization experiments with benzophenone. The
9
time intervals the spectrum exhibits two positive absorption
bands in the 340–470 and 600–750 nm regions and a nega-
tive absorption that corresponds to the ground-state depletion
T
initial absorbance and e for benzophenone triplet were com-
pared with the initial absorbance of the dye. Assuming that the
quantum yield of energy transfer is 1, and using a value of 5800
2
1
21
(
Fig. 3). The signal at 700 nm decays with first-order kinetics
M
cm for the e of benzophenone at 525 nm (17), e of the
T
T
2
1
21
and lifetime of 40–50 ls. Meanwhile, the absorption at 400 nm
does not decay in the same time scale (inset, Fig. 3).
resorufin triplet was found to be 8500 6 2000 M cm at 700
nm, which compares fairly well with that obtained from the blea-
ching experiments. The intersystem crossing quantum yields for
both dyes were obtained by the method of relative actinometry,
using as reference the triplet state of 5,10,15,20-tetraphenyl-21H,
Sensitization experiments were carried out using a thioxanthone
water-soluble derivative (4-[2-hydroxy-3-trimethylaminopropoxy]
thioxanthone), excited with 355 nm laser pulses that excite pre-

3
88 C. Bueno et al.
same and are very much slower for primary amines. A similar
trend between tertiary and primary aliphatic amines was found by
Pietra et al. (11) for the reaction of 2-nitrophenazine 10-oxide.
Furthermore, Sako et al. (12) reported a strong dependence on
the structure of the N-oxide for the photodeoxygenation in the
presence of N,N9-dimethylaniline. The initial bleaching rates
reached a maximum value at 5 mM (inset Fig. 4). At this TEO-
R
HA concentration the photoreaction quantum yield (U ), deter-
mined using Aberchrome as reference, was 0.08.
The photoproduct of the resazurin deoxygenation, resorufin,
also undergoes photodecomposition in the presence of terti-
ary amines. But in this case the only spectral change in the visi-
ble spectrum is the bleaching of the dye. This indicates that the
photoreaction involves the rupture of the dye ring. Primary and
secondary aliphatic amines or anilines do not lead to photocon-
sumption of the dye. The initial bleaching rate, measured at 572
nm, increases with the amine concentration, reaching a maximum
value at 40 mM TEOHA; further amine addition slightly de-
creases the bleaching rate. To get further insight into the photo-
chemical reactions, the quenching of the excited states of the
dyes by several amines was investigated.
Figure 4. Effect of irradiation time on the absorption spectrum of resa-
zurin in the presence of 5 mM TEOHA irradiated at 615 nm in aqueous
solution at pH 9.5. Time from top: 0, 7, 18, 25, 39, 50 min. Inset: effect
of TEOHA concentration on the initial photoreaction rate of resazurin.
2
3H-porphine Zn (ZnTPP). The absorption of the ZnTPP and dye
triplets was measured at several incident laser energies (with opti-
cally matched solutions). From the initial linear part of the plot of
Quenching by amines
absorbance vs laser energy, the product U e for the dye relative to
T T
4
that of ZnTPP was obtained. Using U
M
T
¼ 0.83 and e
cm for ZnTPP (18), and the calculated values of e
dyes, values of UT of 0.08 and 0.04 were obtained for resazurin
and resorufin, respectively. The lower U for resorufin is in agree-
ment with the high radiative deactivation (U
¼ 0.73). On the other
T
¼ 7.3 3 10
Singlet quenching. The fluorescence of the resazurin and resoru-
fin in water at pH 9.5 was quenched by amines, without changes
in the spectral shape. In addition ground-state spectra do not
show any change in the presence of the quencher. Thus, ground-
state complex formation can be disregarded. Bimolecular rate
constants were determined from the Stern–Volmer (SV) plots of
2
1
21
T
for the
T
F
hand, the low intersystem crossing for resazurin is more difficult to
explain. In the absence of reducing agents we did not find loss of
the chromophore, although photorearrangements have been pro-
posed for the singlet excited state of several N-oxides (10). There-
fore, the low intersystem crossing quantum yield can only be
explained in terms of a fast, radiationless deactivation of the singlet
state. The photophysical parameters for the triplet states of both
dyes are also collected and are shown in Table 1.
0
0
0
I /I or s /s vs amine concentration [Q], where I and I stand for
0
the fluorescence intensity, and s and s are the fluorescence life-
times in the absence and the presence of amine, respectively.
0
0
1
0
I =I or s =s ¼ 1 þ KSV½Qꢃ ¼ 1 þ k
q
s ½Qꢃ
ð2Þ
For low quencher concentrations, the Stern–Volmer plots deter-
mined from the fluorescence intensity showed a linear relationship.
But for the stronger quenchers, aromatic amines, steady-state SV
plots showed small positive deviations, indicating some contribu-
tion of static quenching. Nevertheless, in these cases SV plots
based on lifetime measurements were linear, and quenching rate
Photoreactions with amines
The photochemical deoxygenation of N-oxides mediated by
amines has been described for some heterocyclic N-oxides (10).
These data indicate that the characteristics of the process are very
dependent on the structure of the N-oxide and the amine used.
We found that irradiation at 615 nm of resazurin in water at pH
1
q
constants were evaluated from the slopes. The k values for the
different amines were collected and are shown in Table 2. The
values are similar for both dyes. The rate constants approach
a limiting value for amines of low oxidation potential and
decrease when the oxidation potential increases. This is in agree-
ment with the expectation for an electron transfer reaction from
the amine to the excited dye.
9
.5 in the presence of TEOHA changes the solution from blue to
pink. This reaction is not observed in the dark or when the dye
is irradiated alone. The absorption spectra at different irradiation
times are shown in Fig. 4. A new product with a maximum at
5
72 nm is formed, and a well-defined isosbestic point is ob-
Triplet quenching. In the presence of amines a shortening of
the triplet lifetime of the dyes was observed. The bimolecular
quenching rate constants were determined from the experimentally
measured first-order decay (kobs) according to (Eq. 3)
served at 582 nm. The final spectrum corresponds to the deoxy-
genated dye, resorufin, being the only product even at longer
irradiation time. Interestingly, this process only occurs in the
presence of aliphatic tertiary amines. Secondary amines, the bicy-
clic aliphatic amine 1,4-diazabicyclo[2.2.2]octane (DABCO), and
aromatic amines such as aniline or N,N9-dimethylaniline did not
give any photochemical reaction. Primary amines led to the
bleaching of the dye, but the formation of resorufin was not ob-
served. The photoreaction rates (R) were determined from the
plots of absorbance decrease at 615 nm, where the photoproduct
does not absorb, vs time. The initial rates for different tertiary
amines, TEOHA, triethylamine or diethylethanolamine are the
3
kobs ¼ k
0
þ k
q
½Amꢃ
ð3Þ
0
where k is the decay rate constant in the absence of amine. The
3
values of k are included in Table 2. The triplet quenching can
q
be described by the electron transfer process
ꢂ2ꢀ
þ Amꢂþ
D
ð4Þ
ðD Þ þ Am !ðD . . . AmꢂþÞ
ꢀ
ꢁ
ꢂ2ꢀ
DH þ AmðꢀHÞꢂ ð5Þ
ꢂꢀ

Photochemistry and Photobiology, 2002, 76(4) 389
Table 2. Quenching rate constants by amines
1
9
21 21
s
3
7
21 21
s
k
q
3 10 M
k
q
3 10 M
Amine
DABCO
TEOHA
Triethylamine
Di-isobutylamine
Di-isopropylamine
Dipropylamine
Diethylamine
Resazurin
Resorufin
Resazurin
6.2
Resorufin
5.7
3.5
5.7
—
0.9
1.7
0.60
0.3
4.8
4.7
4.0
1.7
0.4
1.5
0.4
0.08
5.7
0.19
0.22
0.12
0.11
0.05
n-butylamine
N,N-dimethylaniline
4.4
Figure 5. Transient absorption spectrum of resazurin in the presence of
0
.5 mM TEOHA in water at pH 9, 40 ls after the laser pulse.
where the in-cage proton transfer takes place in competition with
the ionic dissociation into free radical ions.
parameters is shown as the solid line in the inset in Fig. 4. This
correlation confirms that the deoxygenation reaction proceeds
from the interaction of the triplet state of resazurin with the
amine. Furthermore, the maximum UR (0.08 for 5 mM TEOHA)
is almost equal to the measured intersystem crossing quantum
For both dyes the bimolecular rate constants for the quenching
by aliphatic amines are far from the diffusional limit. Data ob-
tained for the quenching of resorufin correlate with the oxidation
potential of the amine, as expected from a charge transfer
quenching mechanism. The rate constants for triplet quenching
are lower than those of the singlet quenching, in agreement with
that expected for a triplet process. The value of the triplet-
quenching rate constant by TEOHA is much lower for resorufin
than for resazurin. This may be due to the fact that the resorufin
ring, because of the lack of the oxygen atom, is a weaker elec-
tron acceptor than the heterocyclic N-oxide, resazurin.
T
yield. This indicates that a is close to 1. Considering that the
interaction of the resazurin triplet state with the amine involves
an electron transfer mechanism, these results indicate that the
photobleaching takes place after the charge transfer intermediate
formation and leads almost exclusively to the deoxygenated
product. The possible involvement of the singlet state can be dis-
regarded. Although, for amine concentration of up to 20 mM
there is no interaction with the singlet state, experiments carried
out at TEOHA concentrations over 40 mM showed a decrease
in the photoreaction rate that is even higher than that predicted
by Eq. 6. This further confirms that the triplet state is the only
excited state involved in the deoxygenation reaction. Pietra et al.
The transient absorption spectrum of resazurin in the presence
of TEOHA is shown in Fig. 5. This spectrum presents a main
absorption in the region of 400 nm. The band is broader than
that observed in this region for the long-lived absorption arising
in the self-quenching of the dye triplet (Fig. 2). Most likely, the
band in the presence of the amine has contributions from the
protonated and the unprotonated semireduced forms of the dye.
It is interesting to note that the band at 480 nm is absent in
Fig. 5. Therefore, the absorption at 480 nm in the spectrum at 90
ls, shown in Fig. 2, can be assigned to the semioxidized form of
(11) have reported that the photodeoxygenation of 2-nitrophena-
zine 10-oxide in the presence of triethylamine occurs with a quan-
tum yield of 0.2. From the comparison of the fluorescence
quenching, they propose that the photoreaction proceeds through
an exciplex derived from the singlet state.
ꢂ
the dye (D ) produced in reaction 1 (Eq. 1).
The experimental data of the initial bleaching rate of resorufin
as a function of the TEOHA concentration can also be fitted well
with (Eq. 6), indicating that the reaction proceeds exclusively
from the interaction of the triplet dye with the amine. The photo-
bleaching quantum yield measured at 50 mM TEOHA was
A similar behavior was obtained for resorufin. The transient
absorption spectrum in the presence of TEOHA presents long-
lived absorptions in the 400 and 600 nm regions (Fig. 6). This
spectrum is different from that found in the self-quenching of the
resorufin triplet (Fig. 3); the maximum is now at 400 nm and
presents a new band at 600 nm. These bands are ascribed to the
combined absorption of the protonated and the unprotonated
semireduced forms of the dye.
The dependence of the photoreaction of resazurin with the
TEOHA concentration shown in the inset in Fig. 4 indicates that
the photodeoxygenation arises from the triplet state of the dye.
The rate reaches a maximum value at 5 mM TEOHA, where
more than 95% of the triplets are quenched by the amine. If the
reaction proceeds only from the triplet state, the photodeoxy-
genation quantum yield (U
R
) will be given by (Eq. 6)
0
ꢀ
T
3
k
q
½Amꢃ
0
ꢀ
R
¼ 1
a
T
ð6Þ
0
S
ꢀ
1
ꢀ1
k
q
½Amꢃ þ ðs Þ 3k
q
½Amꢃ þ ðs Þ
T
where a
ygenation reaction. The fitting of the experimental data to Eq. 6
T
represents the fraction of triplets that leads to the deox-
Figure 6. Transient absorption spectrum of resorufin in the presence of
50 mM TEOHA in water at pH 9, 40 ls after the laser pulse.
0
0
q T T
using the measured values of k , s , U and a as adjustable

3
90 C. Bueno et al.
0.002, indicating that the photoreduction by the amine is a minor
deactivation process of the resorufin triplet.
4. Burka, L. T., F. P. Guengerich, R. J. Willard and T. L. Macdonald
1985) Mechanism of cytochrome P-450 catalysis. Mechanism of N-
dealkylation and amine oxide deoxygenation. J. Am. Chem. Soc.
07, 2549–2551.
(
An electron transfer mechanism for the photodeoxygenation of
heterocyclic N-oxides in the presence of N,N9-dimethylaniline
was proposed by Sako et al. (12). They suggested that the oxy-
gen loss takes place through an intermediate formed by the re-
combination of the neutral radicals formed by proton transfer
after the initial electron transfer step. A similar mechanism may
be involved in the resazurin–amine photoreaction. This is further
supported by the fact that we found that the photoreaction is
inhibited by the presence of acrylamide. Acrylamide acts as a
radical trap, initiating a chain reaction that leads to polymeri-
zation (14).
1
5
. Albini, A. and M. Alpegiani (1984) The photochemistry of the N-
oxide function. Chem. Rev. 84, 43–71.
. Maki, I., M. Sako, K. Shimada and K. Hitata (1988) Pyrimido
6
[
5,4-g]pteridine N-oxide as a simple chemical model of hepatic mono-
oxygenase. Stud. Org. Chem. 33, 465–469.
7
8
9
. Blanchard, G. J. and C. A. Cihal (1988) Orientational relaxation dy-
namics of oxazine 118 and resorufin in the butanols. Valence- and
state-dependent solvation effects. J. Phys. Chem. 92, 5950–5954.
. Flamigni, L., E. Venuti, N. Camaioni and F. Barigelletti (1989) A
spectroscopic investigation of the temperature and solvent sensitiv-
ities of resorufin. J. Chem. Soc., Faraday Trans. 285, 1935–1943.
. Neumann, M. G., C. C. Schmitt, C. M. Previtali and S. G. Bertolotti
(
1996) Photoreduction of resazurin in the presence of aliphatic
amines. Dye Pigm. 32, 93–99.
CONCLUSIONS
1
1
0. Albini, A., E. Fasani and A. M. Amer (1995) Photochemistry of N-
oxides, pp. 879–891. CRC Handbook of Organic Photochemistry
and Photobiology. CRC Press, Boca Raton, FL.
1. Pietra, S., G. F. Bettinetti, A. Albini, E. Fasani and R. Oberti (1978)
Photoreaction of 2-nitrophenazine 10-oxide with amines. J. Chem.
Soc., Perkin II 185–189.
12. Sako, M., K. Shimada, K. Hirota and Y. Maki (1986) Photochemical
oxygen atom transfer reaction by heterocycle N-oxides involving
a single-transfer process: oxidative demethylation of N,N-dimethyla-
niline. J. Am. Chem. Soc. 108, 6039–6041.
3. Souto-Bachiller, F. A., E. P e´ rez-Inestrosa, R. Suau, R. Rico-G o´ mez,
L. A. Rodr ´ı guez-Rodr ´ı guez and M. E. Coronado-P e´ rez (1999) Photo-
chemistry and photophysics of papaverine N-oxide. Photochem.
Photobiol. 70, 875–881.
Photophysical and photochemical properties of the phenoxazin-
3
-one dyes, resazurin and resorufin, have been quantitatively in-
vestigated in aqueous solution based on the measurements of
absorption and fluorescence spectra, fluorescence lifetimes and
transient absorption. Both dyes are fluorescent only in their an-
ionic form, pH above 7.0. Resorufin presents a very high fluores-
cence quantum yield in neutral and basic media. This makes this
compound a potential target to be used in biological systems.
Singlet and triplet excited states of these dyes are quenched by
amine through an electron-transfer mechanism. But only aliphatic
tertiary amines catalyze the N–O bond cleavage of the N-oxide,
giving resorufin. This photoreaction proceeds from the triplet
after the formation of the charge transfer intermediate with
efficiency close to 1.
1
1
4. Villegas, L., M. V. Encinas, A. M. Rufs, C. Bueno, S. Bertolotti and
C. M. Previtali (2001) Aqueous photopolymerization with visible-
light photoinitiators: acrylamide polymerization photoinitiated with
a phenoxazine dye/amine system. J. Polym. Sci. Part A: Polym.
Chem. 39, 4074–4082.
Acknowledgements—This work was supported by FONDECYT
3010004 and 1000747) and CONICET (Argentina).
15. Heller, H. G. and J. R. Loangan (1981) Photochromic heterocyclic
fulgides. The use of (E)-a-(2,5-dimethyl-3-furylethylide)(isopropyli-
dene)succinic anhydride as a simple convenient chemical actinome-
ter. J. Chem. Soc., Perkin Trans. 2, 341–343.
(
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