Stopped-Flow Kinetics Investigation of the Imidazole-Catalyzed Peroxyoxalate Chemiluminescence Reaction

Article abstract of DOI:10.1021/jo9722109

The stopped-flow technique was used to study the temperature-dependent kinetics of the imidazole- catalyzed peroxyoxalate reaction in order to further elucidate the reaction mechanism. Pseudo- first-order rate constants were obtained from the chemiluminescence intensity vs time profiles for the sequential reaction model X → Y → Z over a wide range of initial concentrations of each of the following reagents: bis(2,4,6-trichlorophenyl) oxalate (TCPO), imidazole (ImH), and hydrogen peroxide. These measurements were complemented by UV absorbance measurements of the kinetics of the step X → Y. For both reaction conditions pseudo-flrst-order in TCPO ([ImH], [H₂O₂] [TCPO]) and pseudo-first-order in H₂O₂ ([ImH][TCPO][H₂O₂]), the first step of the reaction is nucleophilic substitution by two imidazole molecules to form 1,1′-oxalyldiimidazole (ODI). Under conditions of excess TCPO in the concentration range 0.075-0.25 mM, the Y → Z reaction probed the subsequent reaction of ODI with H₂O₂ to form the imidazoyl peracid intermediate, ImC(O)C(O)OOH, For excess H₂O₂ concentrations in the range 2.5-15 mM, the reaction of H₂O₂ with ODI is fast, and the Y → Z step of the sequential reaction model describes subsequent reactions of the imidazoyl peracid. An important unexpected finding necessary for interpreting the kinetics of this reaction is that under conditions of a large excess of H₂O₂ the faster rise of the chemiluminescence signal corresponds to the second step of the reaction (Y → Z), and the slower fall of the signal corresponds to the first step (X → Y). Lutidine and collidine, amine bases of similar aqueous pK_a as imidazole, displayed very little catalytic effect on the PO-CL reaction in comparison to imidazole, corroborating the conclusion that nucleophilic catalysis with formation of ODI as an intermediate constitutes the principal reaction pathway under conditions of both excess oxalate ester and excess H₂O₂. Imidazole quenches the quantum yield of the reaction, a result that can be well explained by catalysis of the decomposition of the key energy-transfer intermediate.

Full text of DOI:10.1021/jo9722109

J . Org. Chem. 1998, 63, 3023-3031
3023
Stop p ed -F low Kin etics In vestiga tion of th e Im id a zole-Ca ta lyzed
P er oxyoxa la te Ch em ilu m in escen ce Rea ction
Andrew G. Hadd, Alex L. Robinson, Kathy L. Rowlen, and J ohn W. Birks*
Department of Chemistry and Biochemistry and Cooperative Institute for Research in Environmental
Sciences (CIRES), Campus Box 216, University of Colorado, Boulder, Colorado 80309-0216
Received December 5, 1997
The stopped-flow technique was used to study the temperature-dependent kinetics of the imidazole-
catalyzed peroxyoxalate reaction in order to further elucidate the reaction mechanism. Pseudo-
first-order rate constants were obtained from the chemiluminescence intensity vs time profiles for
the sequential reaction model X f Y f Z over a wide range of initial concentrations of each of the
following reagents: bis(2,4,6-trichlorophenyl) oxalate (TCPO), imidazole (ImH), and hydrogen
peroxide. These measurements were complemented by UV absorbance measurements of the kinetics
of the step X f Y. For both reaction conditions pseudo-first-order in TCPO ([ImH], [H2O2] .
[TCPO]) and pseudo-first-order in H2O2 ([ImH].[TCPO].[H2O2]), the first step of the reaction is
nucleophilic substitution by two imidazole molecules to form 1,1′-oxalyldiimidazole (ODI). Under
conditions of excess TCPO in the concentration range 0.075-0.25 mM, the Y f Z reaction probed
the subsequent reaction of ODI with H2O2 to form the imidazoyl peracid intermediate, ImC(O)C-
(O)OOH. For excess H2O2 concentrations in the range 2.5-15 mM, the reaction of H2O2 with ODI
is fast, and the Y f Z step of the sequential reaction model describes subsequent reactions of the
imidazoyl peracid. An important unexpected finding necessary for interpreting the kinetics of this
reaction is that under conditions of a large excess of H2O2 the faster rise of the chemiluminescence
signal corresponds to the second step of the reaction (Y f Z), and the slower fall of the signal
corresponds to the first step (X f Y). Lutidine and collidine, amine bases of similar aqueous pKa
as imidazole, displayed very little catalytic effect on the PO-CL reaction in comparison to imidazole,
corroborating the conclusion that nucleophilic catalysis with formation of ODI as an intermediate
constitutes the principal reaction pathway under conditions of both excess oxalate ester and excess
H2O2. Imidazole quenches the quantum yield of the reaction, a result that can be well explained
by catalysis of the decomposition of the key energy-transfer intermediate.
In tr od u ction
kinetics and quantum yield on each of the reagents.11
Therefore, further understanding of the reaction mech-
anism may improve the limits of detection and lead to
the development of new chemiluminescence systems.
Although never isolated despite several kinetics and
mechanistic studies, 1,2-dioxetanedione (I) has frequently
been suggested to be the key intermediate in the PO-CL
reaction.10,12,13
Although the peroxyoxalate chemiluminescence reac-
tion has been successfully used for selective and sensitive
detection of trace analytes,1-3 the mechanism and iden-
tity of the intermediate(s) responsible for the chemilu-
minescence have not been completely elucidated.4-10
Peroxyoxalate chemiluminescence (PO-CL) is described
by the reaction of an aryl oxalate ester with hydrogen
peroxide in the presence of a fluorophor to generate light.
Optimizing PO-CL for analytical detection relies on an
adequate model for the dependence of the reaction
Kinetics studies by Catherall et al. suggest that the
intermediate contains an aryl group (II),5 and Milofsky
and Birks have proposed the formation of a six-membered
intermediate (III) on the basis of their analysis of the
photoinitiated PO-CL reaction in which reaction condi-
tions are pseudo-first-order in oxalate ester.7
Alvarez et al. proposed that multiple intermediates
were capable of generating the observed light in the PO-
CL reaction and that formation of different intermediates
was dependent on the concentration and nature of the
catalyst.6
(1) Robards, K.; Worsfold, P. J . Anal. Chim. Acta 1992, 266, 147-
173.
(2) Hadd, A. G.; Birks, J . W. In Selective Detectors: Environmental,
Industrial, and Biomedical Applications; Sievers, R. E., Ed.; J ohn Wiley
and Sons: New York, 1995; Vol. 131, pp 209-239.
(3) Bowie, A. R.; Sanders, M. G.; Worsfold, P. J . J . Biolumin.
Chemilumin. 1996, 11, 61-90.
(4) Catherall, C. L.; Palmer, T. F.; Cundall, R. B. J . Chem. Soc.,
Faraday Trans. 2 1984, 80, 837-849.
(5) Catherall, C. L. R.; Palmer, T. F.; Cundall, R. B. J . Chem. Soc.,
Faraday Trans. 2 1984, 80, 823-834.
(6) Alvarez, F. J .; Parekh, N. J .; Matuszewski, B.; Givens, R. S.;
Higuchi, T.; Schowen, R. L. J . Am. Chem. Soc. 1986, 108, 6435-6437.
(7) Milofsky, R. E.; Birks, J . W. J . Am. Chem. Soc. 1991, 113, 9715-
9723.
(8) Orlovic, M.; Schowen, R. L.; Givens, R. S.; Alvarez, F.; Matusze-
wski, B.; Parekh, N. J . Org. Chem. 1989, 54, 3605.
(9) Lee, J . H.; Lee, S. Y.; Kim, K.-J . Anal. Chim. Acta 1996, 329,
117-126.
(10) Stevani, C. V.; Lima, K. F.; Toscano, V. G.; Baader, W. J . J .
Chem. Soc., Perkin Trans. 2 1996, 989-995.
(11) Orosz, G.; Givens, R. S.; Schowen, R. L. Crit. Rev. Anal. Chem.
1996, 26, 1-27.
(12) Rauhut, M. M.; Bollyky, L. J .; Roberts, B. G.; Loy, M.; Whitman,
R. H.; Iannotta, A. V.; Semsel, A. M.; Clarke, R. A. J . Am. Chem. Soc.
1967, 89, 6515-6522.
(13) Bollyky, L. J .; Whitman, R. H.; Roberts, B. G.; Rauhut, M. M.
J . Am. Chem. Soc. 1967, 89, 6523-6526.
S0022-3263(97)02210-X CCC: $15.00 © 1998 American Chemical Society
Published on Web 04/14/1998

3024 J . Org. Chem., Vol. 63, No. 9, 1998
Hadd et al.
comparing the time course of the chemiluminescence
emission with changes in the UV absorbance spectrum
for the reactants and products. All of the results obtained
are shown to be consistent with the hypothesis10,15-17 that
the imidazole-catalyzed peroxyoxalate reaction proceeds
through the intermediate ODI.
Hanaoka et al. compared different catalysts for the PO-
CL reaction and found that imidazole (ImH), relative to
other amine bases, had the greatest effect on the chemi-
luminescence yield and kinetics.14 The catalytic effect
of imidazole greatly exceeds predictions based solely on
its basicity and has been attributed to a combination of
nucleophilic and general-base catalysis.8 Recently, other
researchers have suggested that the predominant role
of imidazole in the PO-CL reaction is as a nucleophilic
catalyst, with formation of 1,1′-oxalyldiimidazole (ODI)
as the main precursor in the PO-CL reaction.10,15-17
Exp er im en ta l Section
Ch em ica ls. Bis(2,4,6-trichlorophenyl) oxalate, imidazole,
sodium salicylate, 2,6-dimethylpyridine (lutidine), 2,4,6-trim-
ethylpyridine (collidine), and 9,10-diphenylanthracene (DPA)
were obtained from Aldrich. Hydrogen peroxide (30% in
water), obtained from Mallinckrodt, was titrated with
a
standardized sodium thiosulfate solution to determine the
exact concentration prior to dilution. All other chemicals and
solvents were used without further purification. HPLC-grade
ethyl acetate (Fischer) was the solvent in all experiments.
Reaction mixtures contained small amounts of water origi-
nating in the stock solution of H2O2.
UV Ab sor ba n ce Mea su r em en t s in t h e Ab sen ce of a
F lu or op h or . A Cary 1E UV/vis spectrophotometer was used
to measure the absorbance of 2,4,6-trichlorophenol at 290 nm
formed in the reaction of TCPO and imidazole. In a typical
experiment, 0.5 mL of 0.20 mM TCPO in ethyl acetate from
one syringe was mixed with 0.50 mL of 10.0 mM imidazole in
ethyl acetate from another syringe by manually plunging the
contents of both syringes into a 1.2-mL cuvette placed in the
sample housing of the spectrophotometer. Reported rate
constants were derived from a minimum of three measure-
ments using a nonlinear least-squares fit to a single-exponen-
tial rise expression.20
Ch em ilu m in escen ce Measu r em en ts. Stopped-flow chemi-
luminescence measurements were made using an Applied
Photophysics DX-17M stopped-flow spectrophotometer (Ap-
plied Photophysics, Leatherhead, U.K.), thermostated with a
Neslab RTE-110 cooler. Stock solutions of 2.0 mM TCPO, 2.0
mM DPA, 0.10 M imidazole, and 0.10 M hydrogen peroxide
were prepared in ethyl acetate, stored in the dark, and remade
weekly. In a typical experiment, a solution containing 0.20
mM TCPO and 0.20 mM DPA was prepared and added to one
syringe of the stopped-flow instrument. Mixtures of hydrogen
peroxide, 10 mM, and imidazole, 5.0-40 mM, were added to
the other syringe. The reagents were mixed and profiles of
chemiluminescence intensity versus time obtained after 1.5
ms. To enhance the signal-to-noise ratio, no wavelength
discrimination was used when following the emission of DPA.
Tabulated rate constants reflect the final post-mixing concen-
tration. The chemiluminescence profile area, when ratioed to
the initial concentration of the limiting reagent, is directly
proportional to the quantum yield of the reaction. Profile
areas, which are proportional to chemiluminescence quantum
yields, were used to evaluate the relative effect of different
reagents on the formation of light-generating intermediates.
Our areas are relative to an arbitrary value of 1000 for the
most intense chemiluminescence profile obtained (an entry in
Table 2). Very little additional mechanistic insight would be
provided by absolute quantum yields.
We have studied the kinetics of ODI formation in the
reaction of imidazole with two oxalate esters18 and the
chemiluminescence reaction of ODI with hydrogen per-
oxide in the presence of a fluorophor19 in order to clarify
the role of imidazole catalysis in PO-CL. The rate-
determining step in the formation of ODI from nucleo-
philic substitution of imidazole with bis(2,4,6-trichlo-
rophenyl) oxalate (TCPO) and bis(2,4-dinitrophenyl)
oxalate (DNPO) was found to be dependent on the
structure and nonaqueous pKa of the phenol leaving
group. For TCPO, the rate-determining step was found
to be expulsion of 2,4,6-trichlorophenol (TCP); substitu-
tion of the second phenol with imidazole is much faster
than the first. For DNPO, addition of imidazole to a
carbonyl is the rate-limiting step. The reaction of ODI
with hydrogen peroxide in the presence of a fluorophor
generates light in a reaction analogous to PO-CL. Be-
cause some discrepancy exists as to the number and
nature of the reactive intermediates and the catalytic role
of imidazole, we have investigated the kinetics of the PO-
CL reaction using the stopped-flow technique over a wide
range of relative reactant concentrations. To simplify the
kinetics, all experiments were carried out under pseudo-
first-order reaction conditions with either H2O2 in large
excess over TCPO or TCPO in large excess over H2O2.
Also, ImH was always in large excess over TCPO.
We report for the first time the temperature-dependent
kinetics of the PO-CL reaction using reaction conditions
pseudo-first-order in both the oxalate ester (TCPO) and
hydrogen peroxide. The effects of each of the chemilu-
minescence reagents on the reaction kinetics and on the
relative chemiluminescence quantum yield were deter-
mined. The reaction was evaluated using imidazole,
sodium salicylate, lutidine, and collidine as catalysts in
order to establish the unique catalytic role of imidazole.
The initial steps of the reaction were determined by
An a lysis of Ra te Da ta . The kinetics of each profile was
analyzed using a sequential-reaction model7,8
k′1
9
k′2
9
X
8 Y
8 Z
(1)
where X represents reactants; Y, intermediates; and Z, prod-
ucts; and k′1 and k′2 are pseudo-first-order rate constants.
Using this model, the chemiluminescence intensity is propor-
tional to the concentration of species Y, and both reaction steps
are first-order, irreversible reactions. The integrated rate law
describes the combined exponential contribution of the two rate
constants to the observed signal intensity, I, at any time, t,
(14) Hanaoka, N.; Givens, R. S.; Schowen, R. L.; Kuwana, T. Anal.
Chem. 1988, 60, 2193-2197.
(15) Neuvonen, H. J . Chem. Soc., Perkin Trans. 2 1995, 945.
(16) Stigbrand, M.; Ponten, E.; Irgum, K. Anal. Chem. 1994, 66,
1766.
(17) Emteborg, M.; Ponten, E.; Irgum, K. Anal. Chem. 1997, 69,
2109-2114.
(18) Hadd, A. G.; Birks, J . W. J . Org. Chem. 1996, 61, 2657-2663.
(19) Hadd, A. G.; Seeber, A.; Birks, J . W. Manuscript in preparation.
(20) Espenson, J . H. Chemical Kinetics and Reaction Mechanisms;
McGraw-Hill: New York, 1981.

ImH-Catalyzed Peroxyoxalate Chemiluminescence Reaction
J . Org. Chem., Vol. 63, No. 9, 1998 3025
F igu r e 1. Plot comparing chemiluminescence intensity of the
PO-CL reaction with the UV absorbance of 2,4,6-trichlorophe-
nol vs time in the reaction of 0.10 mM TCPO and 5.0 mM
imidazole. The chemiluminescence intensity (b), left axis, was
measured by adding 0.10 mM DPA and 5.0 mM H2O2. UV
absorbance is shown on the right axis for the reaction without
hydrogen peroxide (2), and with 5.0 mM H2O2 (9).
F igu r e 2. Effect of imidazole concentration on k′1 for the PO-
CL reaction measured by chemiluminescence (b), and the
reaction of 0.10 mM TCPO with imidazole without H2O2 (2)
and with 5.0 mM H2O2 (9) measured by UV absorbance of
2,4,6-trichlorophenol at 290 nm.
TCP is formed, contributing to the higher final absor-
bance at 290 nm. On the basis of our previous study of
the kinetics of the reaction of TCPO with ImH, this
product is ODI.18 The CL signal follows rise-fall kinetics
(eq 1) with a fast increase in the signal followed by a
slower decrease. A nonlinear least-squares fit of the CL
profile to eq 2 produced two rate constants, 3.30((0.01)
× 10-1 s-1 for the rapid increase of the CL signal and
3.61((0.01) × 10-2 s-1 for the slower decay. Thus, the
pseudo-first-order rate constant for the signal decay is
in good agreement with the rate constant for formation
of TCP as measured by UV absorption.
As seen in Figure 2, the rate constants from the
reactions monitored by UV absorbance (with and without
5.0 mM H2O2) and the rate constant derived from decay
of the CL signal all have a second-order dependence on
the imidazole concentration. This result, combined with
the good agreement between the rate coefficients ob-
tained for the production of TCP (and ODI) and the slow
decline of the CL signal, indicates that the slow step of
the chemiluminescent reaction corresponds to the double
substitution reaction of ImH with TCPO to form ODI.
Thus, the pseudo-first-order rate constant k′1 corresponds
to the slow decay of the CL signal, and k′2 corresponds
to the fast rise for conditions of [ImH], [H2O2] . [TCPO].
P O-CL Rea ction Eva lu a ted Usin g Con d ition s of
a La r ge Excess of H2O2 Effect of th e TCP O Con cen -
tr a tion . The TCPO concentration was varied in the
range 0.075-0.25 mM in reactions with 5.0 mM H2O2
and 10.0 mM ImH. The derived rate constants, k′1 and
k′2, and the profile areas are summarized in Table 1.
Within experimental error, both k′1 and k′2 are indepen-
dent of the TCPO concentration, confirming that, under
these conditions, the reaction is pseudo-first order in
TCPO. Over a 3.3-fold increase in the TCPO concentra-
tion, the profile area increased linearly by 3.5-fold. This
result is consistent with TCPO being the limiting reagent
under these reaction conditions.
where M is the maximum intensity. Equation 2 may be fit
Mk′1
2
1
I )
(e-k′ t - e-k′ t
)
(2)
k′2 - k′1
equally well by two expressions in which k′1 and k′2 are
interchanged.10,19,20 A unique solution requires an independent
method of measuring k′1 or k′2. As discussed in the Results,
this was accomplished in separate experiments in which the
appearance of the product 2,4,6-trichlorophenol was monitored
by UV absorbance. Pseudo-first-order rate constants were
derived from a nonlinear least-squares fit to eq 2 using the
software Kaleidagraph. Reported rate constants are an aver-
age of at least three independent measurements and were
determined by nonlinear regression fits to a minimum of 500
data points.
Resu lts a n d Discu ssion
Com p a r ison of Ra te Con sta n ts Der ived fr om UV
Ab sor b a n ce a n d Ch em ilu m in escen ce Mea su r e-
m en ts. The reaction of TCPO and imidazole with or
without hydrogen peroxide produced 2,4,6-trichlorophe-
nol, monitored at its maximum absorbance at 290 nm.
This reaction was studied over the imidazole concentra-
tion range of 2.5-15.0 mM with and without 5.0 mM
H2O2. In Figure 1, the absorbance of TCP versus time
produced in the reaction of 0.10 mM TCPO with 5.0 mM
imidazole with and without H2O2 (right axis) is compared
to a chemiluminescence signal profile (left axis). The
chemiluminescence signal was obtained by detecting the
emitted light from diphenylanthracene (DPA) added to
the reaction of 0.10 mM TCPO, 5.0 mM ImH, and 5.0
mM H2O2.
The two reactions monitored by UV absorbance at 290
nm, one with and one without 5.0 mM H2O2, have similar
absorbance profiles and rate constants. The time course
of the absorbance increase follows a single-exponential
rise for both reactions with an average rate constant of
3.08((0.07) × 10-2 s-1, and the final absorbance corre-
sponds to the release of both phenols from TCPO.
Without hydrogen peroxide, a stable product other than
Effect of Im id a zole Con cen tr a tion a n d Tem p er -
a tu r e. The effect of increasing the imidazole concentra-
tion from 2.5 to 20.0 mM on the PO-CL reaction was
evaluated in the temperature range 6-45 °C. For a given

3026 J . Org. Chem., Vol. 63, No. 9, 1998
Hadd et al.
Ta ble 3. Effect of Tem p er a tu r e on th e Der ived
Th ir d -Or d er Ra te Con sta n t for k′c a n d Secon d -Or d er
Ra te Con sta n t for k′2b w ith H2O2 in La r ge Excess over
TCP O
Ta ble 1. Effect of TCP O Con cen tr a tion on k′1, k′2, a n d
P r ofile Ar ea w ith H2O2 in La r ge Excess over TCP Oa
TCPO, mM
k′1, s-1
k′2 s-1
area, au
0.075
0.10
0.15
0.20
0.25
0.141 ( 0.002
0.123 ( 0.002
0.126 ( 0.002
0.123 ( 0.002
0.098 ( 0.003
1.73 ( 0.01
2.52 ( 0.04
1.48 ( 0.02
1.45 ( 0.02
2.23 ( 0.08
175 ( 10
280 ( 15
360 ( 20
485 ( 25
610 ( 30
T, °C
k1c, M-2 s-1
k2b, M-1 s-1
6
15
25
35
45
1900 ( 200
1420 ( 20
1600 ( 150
1430 ( 10
1200 ( 80
41 ( 5
49 ( 7
75 ( 5
120 ( 4
130 ( 4
a
Reaction conditions: 5.0 mM H2O2, 10.0 mM ImH, 0.10 mM
DPA, 25 °C.
F igu r e 4. Effect of hydrogen peroxide concentration on the
chemiluminescence signal vs time profile. Conditions: (2) 2.5
mM, (b) 5.0 mM, (9) 7.5 mM, (O) 10.0 mM, and (1) 15.0 mM
hydrogen peroxide with 5.0 mM imidazole, 0.10 mM TCPO,
and 0.10 mM DPA at 25 °C.
F igu r e 3. Effect of imidazole concentration on the chemilu-
minescence signal vs time profile. Conditions: (2) 2.5 mM, (b)
5.0 mM, (9) 7.5 mM, (O) 10.0 mM, and (×) 15.0 mM imidazole
with 5.0 mM H2O2, 0.10 mM TCPO, and 0.10 mM DPA at 25
°C.
concentration below 10.0 mM but leveled off at higher
concentrations, reaching a maximum of 0.98 ( 0.02 s-1
at 20.0 mM ImH. A plot of k′2 vs [ImH] evaluated in the
linear range passed through the origin within experi-
mental error, the slope of which produces a second-order
rate constant. Values at each temperature for the third-
order rate constant for reaction k1c and the second-order
rate constant for k2b (where k′2 ) k2a + k2b[ImH]) are
summarized in Table 3. The rate constant k1c has a small
negative activation energy of -7 ( 3 kJ mol-1 (∆Sq ) 38
( 8 J K-1 mol-1, r2 ) 0.71), whereas an activation energy
of 24 ( 3 kJ mol-1 (∆Sq ) 117 ( 9 J K-1 mol-1, r2 ) 0.96)
is calculated from an Arrhenius plot for k2b.
The second-order dependence of k1 on ImH concentra-
tion is consistent with the hypothesis that the first step
of the reaction is ImH catalysis of nucleophilic substitu-
tion of ImH to form ODI from TCPO. The first-order
dependence of k2 on ImH concentration at low ImH
concentrations is consistent with base catalysis by ImH
of a subsequent reaction.
Effect of Hyd r ogen P er oxid e Con cen tr a tion . The
chemiluminescence profiles obtained by varying the large
excess of hydrogen peroxide concentration in the range
2.5-15.0 mM are shown in Figure 4, and the derived
pseudo-first-order rate constants and profile areas are
summarized in Table 4. The area increased significantly,
from 460 ( 25 to 920 ( 45 arbitrary units (au), in the
H2O2 concentration range of 2.5-5.0 mM, remained
constant at 920 ( 45 au in the range 5.0 to 10.0 mM,
and decreased slightly to 810 ( 40 au at 15.0 mM. In
the concentration range 5-15 mM, k′1 was independent
of H2O2 concentration. This was confirmed by monitoring
the formation of TCP by UV absorbance; increasing the
Ta ble 2. Effect of Im id a zole Con cen tr a tion on k′1, k′2,
a n d P r ofile Ar ea w ith H2O2 in La r ge Excess over TCP Oa
[imidazole], mM
102k′1, s-1
k′2, s-1
area, au
2.50
3.50
5.00
7.50
10.0
15.0
20.0
1.04 ( 0.01
1.99 ( 0.02
3.61 ( 0.01
7.98 ( 0.02
13.7 ( 0.1
32.2 ( 0.2
58 ( 2
0.148 ( 0.001
0.232 ( 0.001
0.330 ( 0.001
0.534 ( 0.006
0.66 ( 0.01
1,000 ( 50
875 ( 45
650 ( 35
445 ( 25
370 ( 20
195 ( 10
185 ( 10
0.84 ( 0.02
0.84 ( 0.04
a
Reaction conditions: 5.0 mM H2O2, 0.10 mM TCPO, 0.10 mM
DPA, 25 °C.
temperature, as the ImH concentration increased, the
maximum signal intensity increased, and the time to
reach the maximum signal decreased, as seen in Figure
3 for a temperature of 25 °C. The profile area decreased
6-fold, however, as the imidazole concentration was
increased from 2.5 to 20.0 mM. A plot of the profile area
vs [ImH]-1 produces a straight line having a correlation
coefficient of r2 ) 0.998. At each temperature, the
profiles were similar in shape, and, for a given ImH
concentration, the profile area increased with tempera-
ture. The calculated pseudo-first-order rate constants
and the profile areas obtained at 25 °C are summarized
in Table 2. At each temperature, the rate constant k′1
displayed a second-order dependence on the imidazole
concentration (Figure 2). Terms in the fitted equation
k′1 ) k1a + k1b[ImH] + k1c[ImH]2 ≈ k1c[ImH]2
(3)
were dominated by the second-order component, k1c[ImH]2.
The rate constant, k′2, increased linearly with imidazole

ImH-Catalyzed Peroxyoxalate Chemiluminescence Reaction
J . Org. Chem., Vol. 63, No. 9, 1998 3027
Ta ble 4. Effect of Hyd r ogen P er oxid e on k′1, k′2, a n d
Ta ble 5. Effect of Tem p er a tu r e on k′1 a n d k′2 for th e
P r ofile Ar ea w ith H2O2 in La r ge Excess over TCP Oa
Sod iu m Sa licyla te Ca ta lyzed P O-CL Rea ction a
[H2O2], mM
102k′1, s-1
k′2, s-1
area, au
T, °C
103k′1, s-1
k′2, s-1
2.50
5.00
7.50
10.0
15.0
4.45 ( 0.02
2.62 ( 0.02
2.34 ( 0.02
2.35 ( 0.02
2.70 ( 0.02
0.249 ( 0.004
0.217 ( 0.004
0.286 ( 0.004
0.327 ( 0.004
0.421 ( 0.005
460 ( 25
920 ( 45
920 ( 45
920 ( 45
810 ( 40
10
20
30
38
1.22 ( 0.05
2.11 ( 0.04
3.43 ( 0.01
4.95 ( 0.05
0.9 ( 0.1
4.2 ( 0.4
4.9 ( 0.4
8.3 ( 0.5
a
Reaction conditions: 4.0 mM H2O2, 0.10 mM TCPO, 0.10 mM
a
Reaction conditions: 5.0 mM ImH, 0.10 mM TCPO, 0.10 mM
sodium salicylate, 0.10 mM DPA.
DPA, 25 °C.
Ta ble 6. Effect of Im id a zole on k′1, k′2, a n d P r ofile Ar ea
H2O2 concentration from 2.5 to 15.0 mM in the reaction
of 5.0 mM ImH with 0.10 mM TCPO had no significant
effect on the pseudo-first-order rate constant k′1. Ignor-
ing the data at 2.5 mM H2O2, which appear to be
anomalous (see Figure 4; a possible explanation for this
anomaly is given below), the average value of k′1 is
w ith TCP O in La r ge Excess over H2O2
-1
[imidazole], mM
102k′1, s-1
102k′2, s
area, au
2.50
3.50
5.00
7.50
10.0
15.0
20.0
0.83 ( 0.01
1.66 ( 0.01
3.18 ( 0.02
6.51 ( 0.02
11.1 ( 0.2
25.0 ( 0.2
38.1 ( 0.2
0.50 ( 0.01
0.63 ( 0.01
0.95 ( 0.01
1.56 ( 0.02
2.21 ( 0.05
3.67 ( 0.03
5.40 ( 0.03
10.0 ( 0.5
12.5 ( 0.6
10.0 ( 0.5
8.3 ( 0.4
7.0 ( 0.4
4.3 ( 0.2
4.2 ( 0.2
2.5((0.2) × 10-2 s-1
. At 45 mM H2O2, however, an
increase to 5.7(( 0.1) × 10-2 s-1 for k′1 was observed. The
lack of dependence of k′1 on excess H2O2 concentration
in the range 5-15 mM H2O2 is consistent with the first
step of the reaction being the double ImH substitution
to form ODI from TCPO. One possible explanation for
the increased value of k′1 at very high H2O2 concentration
is an additional reaction of the singly substituted TCPO
molecule with H2O2 to form the corresponding peroxy-
oxalate.
a
Reaction Conditions: 5.0 µM H2O2, 0.10 mM TCPO, 0.10 mM
DPA, 25 °C.
Effect of Gen er a l Ba se Ca ta lysts. The PO-CL
reaction was evaluated using two sterically hindered
bases, lutidine and collidine, which have a similar
aqueous pKa as imidazole. Neither base significantly
catalyzed the PO-CL reaction. For equal concentrations
of amine, 2% of the profile area in the imidazole-catalyzed
reaction was obtained with lutidine and 0.6% with
collidine. For 5.0 mM amine, the values for k′1 using
collidine, 9.3 × 10-4 s-1, and lutidine, 1.6 × 10-4 s-1, were,
respectively, 35 and 200 times less than k′1 using imi-
dazole as the catalyst.
The PO-CL reaction catalyzed by sodium salicylate was
evaluated in the 10-38 °C temperature range. In these
experiments, the post-mixing reagent concentrations
were 0.10 mM TCPO, 0.05 mM DPA, 4.0 mM H2O2, and
0.10 mM sodium salicylate. Rate constants, k′1 for the
slow signal decay, and k′2, for the rapid signal increase,
were measured as a function of temperature and are
given in Table 5. Arrhenius plots produce activation
energies of 36.5 ( 0.4 kJ mol-1 (∆Sq ) 188 ( 1 J K-1
mol-1, r2 ) 0.9998) for k′1 and 54 ( 14 kJ mol-1 (∆Sq )
192 ( 48 J K-1 mol-1, r2 ) 0.88) for k′2.
P O-CL Rea ction Eva lu a ted Usin g a La r ge Excess
of TCP O (P seu d o-F ir st-Or d er in H2O2). Effect of
Tem p er a tu r e a n d Im id a zole Con cen tr a tion . The
PO-CL reaction was investigated using conditions in
which the TCPO concentration, 0.10 mM, was in large
excess over the hydrogen peroxide concentration, 5.0 µM,
for imidazole concentrations in the range 2.5-15.0 mM
and over the temperature range 7-45 °C. Qualitatively,
the shape of each chemiluminescence profile was similar
to that shown in Figure 3 with the maximum intensity
increasing and time to reach maximum intensity de-
creasing with increasing imidazole concentration. The
derived rate constants, k′1 and k′2, obtained at 25 °C,
along with the relative profile areas, are summarized in
Table 6. Comparing the profile areas in Tables 2 and 6,
it may be seen that the quantum yield of the reaction is
nearly equal for both reaction conditions after correction
is made for the concentration of the limiting reagent.
In contrast to pseudo-first-order TCPO conditions,
when imidazole is in large excess over TCPO and both
are in excess over H2O2, k′1 reflects the increase of the
CL signal and k′2 the decrease. We infer this by compar-
With the exception of the result obtained at 2.5 mM,
which again appears to be anomalous, the fall rate
constant increased with increasing hydrogen peroxide
concentration. A plot of k′2 versus [H2O2] for all rate
constants given in Table 4 has a positive intercept of 0.18
( 0.04 s-1 and a slope of 16 ( 3 M-1 s-1. Ignoring the
data point at 2.5 mM H2O2, the intercept is 0.13 ( 0.01
s-1 and the slope is 20 ( 1 M-1 s-1
.
The intercept
represents a first-order rate constant for a reaction zero-
order in hydrogen peroxide concentration, and the slope
represents a second-order rate constant for a reaction
first-order in hydrogen peroxide.
The first-order dependence of k′2 on H2O2 concentration
would be consistent with the hypothesis that the second
step of the reaction is substitution of H2O2 for ImH in
ODI. However, this would not explain the large zero-
order component of the dependence of k′2 on [H2O2]. It
is more likely that the first substitution of ODI by H2O2
is very fast (0.6-3.9 s-1 for 2.5-15 mM H2O2 in aceto-
nitrile)19 compared to the conversion of TCPO to ODI, at
least at higher H2O2 concentrations. This is supported
by the UV absorbance experiment shown in Figure 1 in
which the formation of products proceeds at the same
rate in the presence and absence of 5.0 mM H2O2. Thus,
the rate constant k′2 must describe the fate of a product
of the reaction of H2O2 with ODI. As discussed below,
the zero-order component could be due to the unimolecu-
lar cyclization of the intermediate imidazoyl hydroper-
oxide, ImC(O)C(O)OOH, to form an energy-transfer
intermediate, and the first-order dependence of k′2 on
[H2O2] may be due to a second substitution of H2O2 to
form HOOC(O)C(O)OOH. The assumption that the
reaction of H2O2 with ODI is fast compared to the
formation of ODI is consistent with the rate constant
derived below for the H2O2 + ODI reaction under condi-
tions of a large excess of TCPO except in the range of
2.5-5.0 mM H2O2 where the anomalous results were
obtained. Thus, the anomalous effects on k′1 and k′2 at
low H2O2 may be due to the lack of a single, rate-
determining step in this concentration regime.

3028 J . Org. Chem., Vol. 63, No. 9, 1998
Hadd et al.
Ta ble 7. Effect of TCP O Con cen tr a tion on k′1, k′2, a n d
P r ofile Ar ea w ith TCP O in La r ge Excess over H2O2
ing measured rise and fall rate constants and from the
dependencies of the rates on ImH concentration. The CL
signal rises with a second-order dependence on the
imidazole concentration; the corresponding third-order
rate constant of 1.10((0.05) × 102 M-2 s-1 differs by only
30% from the value of 1.60((0.012) × 102 M-2 s-1
obtained for k′1 (CL signal decline) under excess H2O2
conditions. This agreement in rate constants is remark-
able, especially considering the several orders of magni-
tude difference in reagent concentrations; the 30% slower
reaction under excess H2O2 conditions could be attributed
to the higher concentration of water (0.6 to 3.4 mM H2O
for excess H2O2 conditions vs 1.1 µM H2O for excess
TCPO). It is well-known that water strongly affects the
kinetics and quantum yield of the peroxyoxalate reac-
tion.2 The fall of the signal is approximately first-order
in ImH with only a slight second-order contribution.
Thus, under conditions of excess TCPO, the reaction
profile assumes the more familiar situation where the
increase of the chemiluminescence signal follows the
conversion of TCPO to an intermediate(s), and the decay
of that intermediate is slower than its formation.
The pseudo-first-order rate constants for k′1 and k′2
were resolved into zero-, first-, and second-order contri-
butions as described by eqs 4 and 5:
a
[TCPO], mM
k′1, s-1
k′2, s-1
area, au
0.075
0.10
0.15
0.20
0.25
0.115 ( 0.005
0.121 ( 0.003
0.098 ( 0.005
0.115 ( 0.008
0.110 ( 0.003
0.018 ( 0.005
0.025 ( 0.003
0.048 ( 0.004
0.060 ( 0.006
0.081 ( 0.003
15.8 ( 0.8
15.0 ( 0.8
10.0 ( 0.5
15.8 ( 0.8
11.7 ( 0.6
a
Reaction conditions: 5.0 µM ImH, 10.0 mM ImH, 0.10 mM
DPA, 25 °C.
k′1 ) k1d + k1e[ImH] + k1f[ImH]2
k′2 ) k2d + k2e[ImH] + k2f[ImH]2
(4)
(5)
F igu r e 5. Effect of TCPO concentration on the chemilumi-
nescence signal vs time profile and k′2 (inset), with the TCPO
concentration in large excess over the H2O2 concentration.
Conditions: (2) 0.075 mM, (b) 0.10 mM, (9) 0.15 mM, (O) 0.20
mM, and (×) 0.25 mM TCPO with 10.0 mM imidazole, 5.0 µM
H2O2, and 0.10 mM DPA at 25 °C.
The values for k1f in units of M-2 s-1 at 7, 15, 25, 35,
and 45 °C are 700, 840, 1100, 1250, and 1600 s-1
respectively, with an average uncertainty of 100 M-2 s-1
,
.
The activation energy associated with the increase of k1f
with temperature is 15.8 ( 0.8 kJ mol-1 (∆Sq ) 111 ( 3
J K-1 mol-1, r2 ) 0.992).
a plot of the increase in k′2 with increasing TCPO
concentration (inset) are shown in Figure 5. The maxi-
mum signal increased in the concentration range 0.075-
0.20 mM and then began to decrease at 0.25 mM TCPO.
The profile area was nearly constant at ∼15 au, decreas-
ing to ∼12 au at 0.25 mM TCPO. The rate constant k′1
was independent of the TCPO concentration, whereas a
plot of k′2 versus TCPO concentration (inset of Figure 5)
was linear with an intercept not significantly different
Although showing a slight second-order dependence on
imidazole concentration, the value for k′2 was mainly
determined by k2e, the component of the reaction rate that
is first-order in imidazole concentration. For k′2, the
second-order dependence on the imidazole concentration
was unobservable at 7 and 15 °C, and k2f was only 50 (
8 M-2 s-1 at 25 °C and 88 ( 1 M-2 s-1 at 45 °C. The
values for k2e at 7, 15, 25, 35, and 45 °C are 1.2, 2.0, 1.6,
1.6, and 1.2 M-1 s-1 with an average uncertainty of 0.2
M-1 s-1. Thus, the rate constant k2e is approximately
independent of temperature.
The second-order dependence of k′1 on ImH again is
consistent with the first step of the reaction being ImH
catalysis of the nucleophilic substitution of ImH into
TCPO to form ODI. The predominantly first-order
dependence of k′2 on [ImH] is consistent with general
base catalysis by ImH of a subsequent reaction.
As found for conditions of excess H2O2, increasing
concentration of imidazole caused a decrease in profile
area (see Table 6), which is proportional to the chemi-
luminescence quantum yield. Again, the profile area was
inversely related to imidazole concentration.
from zero and slope of 3.7((0.2) × 102 M-1 s-1
.
The lack of dependence of k1 on TCPO concentration
confirms the establishment of pseudo-first-order condi-
tions in TCPO with respect to its reaction with ImH and
further verifies the assignment of k1 to the CL signal rise.
The first-order dependence of k2 on TCPO is well ex-
plained if the second step of the reaction is the nucleo-
philic reaction of H2O2 with ODI since the concentration
of ODI formed in the first step is equal to the initial
TCPO concentration.
Rea ction Mech a n ism
A mechanism consistent with all of the kinetics obser-
vations is given in Scheme 1. Table 8 summarizes the
reaction steps probed by the kinetics and the rate laws
that apply to this mechanism for pseudo-first-order
reaction conditions with either H2O2 or TCPO in large
excess.
Effect of TCP O Con cen tr a tion . The TCPO concen-
tration was varied in the range 0.075-0.25 mM in order
to determine the order of the reaction with respect to
TCPO. Final concentrations of 10 mM ImH and 5.0 µM
H2O2 were used for all experiments. Table 7 gives the
derived values of k′1 and k′2 and relative profile areas.
Examples of the chemiluminescence profiles along with
Excess H2O2 Con d ition s. All of the results are
consistent with the conclusion that under conditions of

ImH-Catalyzed Peroxyoxalate Chemiluminescence Reaction
J . Org. Chem., Vol. 63, No. 9, 1998 3029
reaction of ImH with TCPO.18 The accelerated addition
of the second imidazole apparently results from activation
of the adjacent oxalate carbonyl by the first substituted
imidazole.18
Sch em e 1
ODI has a very short lifetime in the presence of excess
hydrogen peroxide. As seen in Figure 1, the absorbance
profile at 290 nm for the reaction of 0.10 mM TCPO and
5.0 mM ImH has the same first-order rate constant but
a greater absorbance (due to the ODI product) than the
same reaction with 5.0 mM H2O2 added. The similarity
of the rate constants and the lack of a rise-fall absorbance
profile suggests that reaction of ODI with H2O2 is fast
relative to formation of ODI. Thus, the rise rate reaction
described by k′1 results in the formation of an imidazoyl
peracid, species C of Scheme 1, a reaction step commonly
proposed in other mechanistic studies of the PO-CL
reaction.5,7,8,10,12
It was found that hydrogen peroxide does affect the
rise rate at significantly higher concentrations. For
example, at 45 mM H2O2, k′1 is 5.7((0.1) × 10-2 s-1
,
compared to the average 2.5((0.2) × 10-2 s-1 in the 5 to
15 mM range. A zero-order dependence at low H2O2
concentration with a first-order dependence at higher
H2O2 concentration has been observed previously.5,27 At
higher H2O2 concentration, it is possible that H2O2
substitutes directly into TCPO as a result of base
catalysis alone to form the peroxyoxalate intermediate.
The chemiluminescence profile shapes are well ex-
plained if the signal is proportional to the formation and
decay of the imidazoyl peracid, C. Under conditions of a
large excess of H2O2, the decay of C has a complex
dependence on the imidazole concentration with a zero-
and first-order dependence on the hydrogen peroxide
concentration. A plot of k′2 vs [H2O2] is linear, but with
a significantly large, positive intercept. The dependence
of k′2 on these reagents can be described by a mechanism
in which cyclization of C to form a high-energy interme-
diate, E, competes with the base-catalyzed substitution
of an additional molecule of hydrogen peroxide to form
species D of Scheme 1.
a large excess of H2O2 and ImH over TCPO formation of
ODI is the rate-limiting step for the rise reaction. The
pseudo-first-order rate constant, k′1, has a second-order
dependence on the ImH concentration and no dependence
on the initial H2O2 concentration, as measured either by
chemiluminescence or by UV absorbance of the product
TCP. In addition, the absorbance of TCP increased with
one observed rate constant to a final absorbance corre-
sponding to the production of 2 mol of phenol for every
mole of TCPO. These results are consistent with the
nucleophilic substitution of two molecules of imidazole
into TCPO to form ODI:
We propose that cyclization of C to form imidazoylhy-
droxydioxetanone, E, is the primary reaction leading to
chemiluminescence under excess H2O2 conditions with
an estimated unimolecular rate constant of 0.14 s-1
,
The first- and second-order dependence on the imida-
zole concentration is characteristic of nucleophilic sub-
stitution reactions of imidazole in which substitution
by imidazole is catalyzed by another molecule of
imidazole.15,21-23 Ester aminolysis in aprotic solvents is
rate limited by the collapse of the tetrahedral intermedi-
ate.24 For reactions involving imidazole, collapse of the
tetrahedral intermediate is catalyzed by another mol-
ecule of imidazole, either by proton transfer from the
substituted imidazole or proton transfer to the phenol
leaving group.22,25,26 In the PO-CL reaction, the substitu-
tion of the second imidazole to TCPO was apparently fast
in comparison to the first, an observation consistent with
other studies of the reaction of imidazole with oxalate
esters10 and our previous study of the nucleophilic
derived from the intercept of the plot of k′2 versus
hydrogen peroxide concentration. Stevani et al. and
Hohman et al. have demonstrated that aryl monoper-
oxyoxalates, similar to C, are stable in the presence of a
fluorophor, but addition of a base leads to rapid chemi-
luminescence.28,29 Using sodium salicylate, a stronger
base than imidazole, the conversion of C to a high-energy
intermediate is nearly 1 order of magnitude faster,
whereas the formation rate is nearly 1 order of magnitude
slower. This result is consistent with nucleophilic ca-
talysis for the first substitution of hydrogen peroxide and
cyclization of the peroxyoxalate product, leading to the
phenol-substituted hydroxydioxetanone analogue of E
(structure II of the Introduction), as first suggested by
Catherall et al.5 This structure is analogous to dioxet-
anones that have been shown to produce the singlet
excited state of fluorescent acceptor molecules in a
(21) Bruice, T. C.; Schmir, G. L. J . Am. Chem. Soc. 1957, 79, 1663-
1667.
(22) Kirsch, J . F.; J encks, W. P. J . Am. Chem. Soc. 1964, 86, 833-
837.
(23) Kirsch, J . F.; J encks, W. P. J . Am. Chem. Soc. 1964, 86, 837.
(24) Menger, F. M.; Vitale, A. C. J . Am. Chem. Soc. 1973, 95, 4931.
(25) Satterthwait, A. C.; J encks, W. P. J . Am. Chem. Soc. 1974, 96,
7018-7031.
(27) Orosz, G. Tetrahedron 1989, 45, 3493-3506.
(28) Stevani, C. V.; Campos, I. P. d. A.; Baader, W. J . J . Chem. Soc.,
Perkin Trans. 2 1996, 1645-1648.
(29) Hohman, J . R.; Givens, R. S.; Orosz, G. Tetrahedron Lett. 1996,
37, 8273.
(26) Neuvonen, H. J . Chem. Soc., Perkin Trans. 2 1995, 951.

3030 J . Org. Chem., Vol. 63, No. 9, 1998
Ta ble 8. Su m m a r y of P r op osed Rea ction Mech a n ism a n d Ap p r oxim a te Ra te La w s
Hadd et al.
reaction step
excess H2O2
excess TCPO
TCPO + 2ImH f B + 2ArOH
rate ) k1[TCPO][ImH]2
rate ) k1[TCPO][ImH]2
k′1 ) k′rise
k′1 ) k′fall
B + H2O2 f C + ImH
C + H2O2 f D + ImH
C f E
fast except for [H2O2] < 5.0 mM
rate ) k2[B][ImH][H2O2]
k′2 ) k′fall
rate ) k2x[C][ImH][H2O2]
k2x obtained from slope of krise vs [H2O2]
rate ) k2y[C][ImH]
slow, not probed by kinetics
fast, not probed by kinetics
k2y obtained from intercept of krise vs [H2O2]
fast compared to k′1 and k′2
faster than E + F and accounts
for decreasing quantum yield
with increasing [ImH]
E + F f hν + products
E + ImH f non-CL products
fast compared to k′1 and k′2
faster than E + F and accounts
for decreasing quantum yield
with increasing [ImH]
chemically initiated electron-exchange luminescence
(CIEEL) reaction.30-32
concentration. As the imidazole concentration was in-
creased, k′2 increased linearly at low imidazole concen-
tration but reached a maximum above 15 mM ImH. This
dependence on the imidazole concentration can be ex-
plained by a two-step reaction in which hydrogen per-
oxide addition becomes rate-limiting. A similar maxi-
mum pseudo-first-order rate constant was observed in
the imidazole-catalyzed reaction of ODI with hydrogen
peroxide in acetonitrile, and the quantum yield decreased
linearly with increasing H2O2 concentration.19 In con-
trast, in this work only a small reduction of the quantum
yield was observed beyond 10 mM H2O2, suggesting that
in ethyl acetate D might also cyclize to produce an
energy-transfer intermediate.
Excess TCP O Con d ition s. Under conditions of
[ImH] . [TCPO] . [H2O2], k′1 has a second-order
dependence on the imidazole concentration, with a ter-
molecular rate constant of 1.1((0.2) × 102 M-2 s-1. The
nearly identical rate constant and same dependence on
imidazole concentration strongly suggests that the initial
reaction step is similar to what occurs under excess H2O2
conditions. However, in this case the excess reagent,
TCPO (0.075-0.25 mM), is 1-2 orders of magnitude less
than the concentration of H2O2 (2.5-15 mM) under excess
H2O2 conditions. As a result, the data suggest that
reaction of H2O2 with ODI to form the imidazoyl peracid
(C) is sufficiently slow to correspond to the decline of the
chemiluminescence signal. The first-order dependence
of k′2 on the initial TCPO concentration is readily
explained by the reaction of H2O2 with ODI derived from
TCPO. Since loss reactions of C are fast compared to its
formation, the kinetics under excess TCPO conditions
provides no information about subsequent reactions of
C. Those reactions are explored in another paper,19
however, where the starting reagent is ODI rather than
TCPO.
It is possible that the substituted dioxetanone, E, will
release imidazole to form 1,2-dioxetanedione. This reac-
tion may be catalyzed by proton addition to N-3 of the
substituted imidazole from another molecule of imidazole
and collapse of the tetrahedral intermediate.
Bifunctional catalysis by imidazole is well known,33,34
and ester aminolysis reactions in aprotic solvents are
accelerated by general acid catalysis.24 In the case of
3-imidazoyl-3-hydroxydioxetanone, imidazole is an excel-
lent electron-withdrawing leaving group, suggesting that
the lifetime of E is short.
Obviously, an unambiguous assignment of the struc-
ture of the reactive intermediate that leads to chemilu-
minescence cannot be made from analysis of the reaction
kinetics alone. Recently, Stevani et al. examined the
imidazole-catalyzed PO-CL reaction and, on the basis of
previous work with unstable four-membered cyclic per-
oxides, quantum yields of other dioxetanes, and the short-
lived interconversion reactions of carboxylic acids with
good leaving groups, proposed 1,2-dioxetanedione (F ) as
the most likely intermediate in peroxyoxalate chemilu-
minescence.10 Although the results presented in this
study are unable to distinguish between these two
intermediates, we favor E because of the significant
decrease in the quantum yield of the reaction with added
imidazole. That imidazole decreases the quantum yield
of the reaction suggests that deprotonation and formation
of dioxetanedione (or two molecules of CO2 directly) may
be the main cause of signal reduction in the PO-CL
reaction.
Although useful as a diagnostic, rise-fall kinetics as
described by eqs 1 and 2 does not apply exactly to the
proposed mechanism in which excess reagent TCPO is
converted to ODI and followed by a pseudo-first-order
reaction of H2O2. The appropriate integrated rate equa-
tions may be derived for the following mechanism:
Base-catalyzed addition of hydrogen peroxide to imi-
dazoyl hydroperoxide, D, apparently competes with
formation of E and at high H2O2 concentration and may
also reduce the quantum yield of the reaction. The
addition of hydrogen peroxide to imidazoyl hydroperoxy-
oxalate forming dihydroperoxyoxalate, species D in
Scheme 1, has a complex dependence on the imidazole
TCPO + 2ImH f ODI + 2TCP
H2O2 + ODI f C
k1
k2
k3
(8)
(9)
(30) Adam, W.; Liu, J .-C. J . Am. Chem. Soc. 1972, 94, 2894-2895.
(31) McCapra, F. Prog. Org. Chem. 1973, 8, 231.
(32) Schuster, G. B.; Horn, K. A. In Chemical and Biological
Generation of Excited States; Academic Press: New York, 1982; pp
229-247.
C f products + light
(10)
(33) Bender, M. L.; Bergeron, R. J .; Komiyama, M. The Bioorganic
Chemistry of Enzyme Catalysis; Wiley-Interscience: New York, 1984.
(34) Seoud, O. A. E.; Menegheli, P.; Pires, P. A. R.; Kiyan, N. Z. J .
Phys. Org. Chem. 1994, 7, 431-436.
Under conditions of [ImH] . [TCPO] . [H2O2], and
reaction 9 slow compared to reaction 8, the ODI concen-

ImH-Catalyzed Peroxyoxalate Chemiluminescence Reaction
tration is given by
J . Org. Chem., Vol. 63, No. 9, 1998 3031
energy-transfer intermediate as well, the chemilumines-
cence quantum yield is given by
[ODI] ) (1 - e-k t)[TCPO]0
(11)
1
k4a[F]
k3b
φ )
(19)
where k1 is the pseudo-first-order rate constant for a
given imidazole concentration. The differential rate law
for [H2O2] is
(
)(
)
k4a[F] + k4b[ImH] k3b + k3a[H2O2]
Light emission from the peroxyoxalate chemilumines-
cence reaction is known to be linear over several orders
of magnitude change in fluorophor concentration, requir-
ing that k4b[ImH] . k4a[F], and thus, the quantum yield
is predicted to vary inversely with [ImH], as is observed.
If D also cyclizes to form an energy transfer intermediate,
ImH could react with that species as well, again reducing
the quantum yield of the reaction. This model provides
a good description of the effects of all reagents on the
chemiluminescence profile shapes and areas obtained
under conditions of excess ODI concentration.
d[H2O2]
) -k2[H2O2][ODI] ) -k2(1 - e-k t)[TCPO]0
2
dt
[H2O2] (12)
Rearranging and integrating gives a complex time de-
pendence for H2O2 concentration,
t[TCPO]
- (k2/k1)e-k
1
0
[H2O2] ) [H2O2]0e-k [TCPO]0te
(13)
2
If the reaction of C to form products is fast compared to
its formation, we can assume steady state for C and
calculate C from the above expressions for [ODI] and
[H2O2]
Con clu sion s
k
Over a very wide range of reaction conditions, the
kinetics of the imidazole-catalyzed peroxyoxalate reaction
is consistent with the first step of the reaction being
conversion of TCPO to ODI and the second step being
the reaction of H2O2 with ODI to form the imidazoyl
peracid, C. Under conditions of excess H2O2, intermedi-
ate C may either cyclize in a unimolecular reaction to
form a high-energy intermediate, E, or react with an
additional molecule of H2O2 to form D. Imidazole cata-
lyzes all reaction steps with reaction orders consistent
with this mechanism. Imidazole also decreases the
quantum yield of the reaction. This negative effect on
the chemiluminescence efficiency could be accounted for
by catalysis of the decomposition of C to form nonlight
producing products, possibly including 1,2-dioxetanedi-
one, a postulated species long believed to be the key
energy-transfer intermediate in the reaction.
[C]ss
)
2[H2O2][ODI]
(14)
k3
The chemiluminescence signal is then proportional to
[C]ss. In the limit of the conversion of TCPO to ODI being
fast in comparison to the reaction of H2O2 with ODI, the
above kinetics expressions simplify to describe simple
rise-fall kinetics with k1 corresponding to the formation
of ODI and k2 to the reaction of H2O2 with ODI to form
C. The effect of ImH on the quantum yield (profile area)
can be accounted for by the reactions
C + H2O2 f D
C f E
k3a
k3b
(15)
(16)
E + F f light
k4a
k4b
(17)
(18)
Ack n ow led gm en t. The authors thank Shelley Co-
pley for helpful discussions. The stopped-flow instru-
ment was purchased under NSF Grant No. BIR-9302748.
E + ImH f non-CL products
where F is the fluorophor. Assuming that D is not an
J O9722109