Electron exchange luminescence of spiroadamantane-substituted dioxetanes triggered by alkaline phosphatases. Kinetics and elucidation of pH effects

Article abstract of DOI:10.1021/ja961904g

Intramolecular chemically initiated electron exchange luminescence (CIEEL) was studied in the alkaline phosphatase triggering of the spiroadamantyl-substituted 1,2-dioxetanes AMPPD and CSPD, which are widely employed in modern chemiluminescent bioassays, particularly in clinical immunoassay applications. Experimental data on the pH dependence of the CIEEL efficiency, the CIEEL emitter fluorescence lifetime, the rate of cleavage of the dephosphorylated dioxetane 2a, the turnover number, and the Michaelis constant are reported and kinetically rationalized. Through this detailed kinetic analysis, the pH effects on the various steps of the CIEEL process were elucidated. Although every step of the triggering involves H⁺ or HO^- ions, it was shown that at alkaline pH and steady-state conditions only enzymatic dephosphorylation of AMPPD and CSPD depends on pH in this CIEEL process. The turnover number is affected only at pH below 9, which reflects a switchover of the rate-determining step of the enzyme dephosphorylation to its phosphorylation at increasing pH. The Michaelis constant depends on pH within the entire range used, which is attributed to the pH effect on the concentration of the catalytically active enzyme form. The present model kinetic studies on the important alkaline-phosphatase-triggered CIEEL reaction provide the necessary mechanistic insights to understand pH effects in such enzymatic processes for the rational design of more effective CIEEL systems.

Full text of DOI:10.1021/ja961904g

10400
J. Am. Chem. Soc. 1996, 118, 10400-10407
Electron Exchange Luminescence of
Spiroadamantane-Substituted Dioxetanes Triggered by Alkaline
Phosphatase. Kinetics and Elucidation of pH Effects
Waldemar Adam,† Irena Bronstein,‡ Brooks Edwards,‡ Thomas Engel,§
Dirk Reinhardt,† Friedemann W. Schneider,§ Alexei V. Trofimov,*,†,| and
Rostislav F. Vasil’ev|
Contribution from the Institute of Organic Chemistry, UniVersity of Wu¨rzburg, Am Hubland,
D-97074 Wu¨rzburg, Germany, Tropix, Inc., 47 Wiggins AVenue, Bedford, Massachusetts 01730,
Institute of Physical Chemistry, UniVersity of Wu¨rzburg, Marcusstrasse 11, D-97070 Wu¨rzburg,
Germany, and Institute of Biochemical Physics, United Institute of Chemical Physics, Russian
Academy of Sciences, ul. Kosygina 4, Moscow 117977, Russia
ReceiVed June 6, 1996X
Abstract: Intramolecular chemically initiated electron exchange luminescence (CIEEL) was studied in the alkaline
phosphatase triggering of the spiroadamantyl-substituted 1,2-dioxetanes AMPPD and CSPD, which are widely
employed in modern chemiluminescent bioassays, particularly in clinical immunoassay applications. Experimental
data on the pH dependence of the CIEEL efficiency, the CIEEL emitter fluorescence lifetime, the rate of cleavage
of the dephosphorylated dioxetane 2a, the turnover number, and the Michaelis constant are reported and kinetically
rationalized. Through this detailed kinetic analysis, the pH effects on the various steps of the CIEEL process were
elucidated. Although every step of the triggering involves H+ or HO- ions, it was shown that at alkaline pH and
steady-state conditions only enzymatic dephosphorylation of AMPPD and CSPD depends on pH in this CIEEL
process. The turnover number is affected only at pH below 9, which reflects a switchover of the rate-determining
step of the enzyme dephosphorylation to its phosphorylation at increasing pH. The Michaelis constant depends on
pH within the entire range used, which is attributed to the pH effect on the concentration of the catalytically active
enzyme form. The present model kinetic studies on the important alkaline-phosphatase-triggered CIEEL reaction
provide the necessary mechanistic insights to understand pH effects in such enzymatic processes for the rational
design of more effective CIEEL systems.
Introduction
yield of the excited reaction product, the CIEEL emitter) and
the fluorescence efficiency Φfl of the CIEEL emitter.
The chemiluminescence properties of dioxetanes as high-
energy peroxides are of particular interest for the generation of
excited states without light. The formation of electronically
excited products can be induced either thermally or by an
electron-transfer mechanism, originally discovered by Schuster
for the diphenoyl peroxide1 and in the meantime abundantly
documented for the R-peroxy lactones2 and appropriate dioxe-
tanes.3 This phenomenon of light emission derived from
electron transfer chemistry is known as chemically initiated
electron exchange luminescence (CIEEL). At the reaction rate
V, intensity of the CIEEL iCIEEL may be expressed by eq 1, in
which ΦCIEEL ) Φ*Φfl is the CIEEL yield
The CIEEL may result from both intermolecular and in-
tramolecular electron transfer. The latter case has been at-
tributed, in particular, to the firefly luciferase-luciferin biolu-
minescence.4 For this intramolecular CIEEL process the
chemiluminescence quantum yield is over 90% in ViVo, i.e.,
chemical energy is efficiently converted into electronic excitation
manifested by light emission.
Also 1,2-dioxetanes with substituents of low oxidation
potentials such as the aryl-O- functionality display intramo-
lecular CIEEL. The chemiexcitation step consists of the
cleavage of the intermediate dioxetane phenolate anion. Such
cleavage is initiated by the intramolecular electron transfer from
the oxidizable phenoxide functionality to the antibonding σ*
orbital of the peroxide bond. These phenolate-initiated intramo-
lecular CIEEL processes provide the basis for numerous
commercial applications, most prominently in chemiluminescent
immunoassays. Numerous research efforts were undertaken to
develop more efficient CIEEL systems for the above purposes.
The most successful design5,6 utilizes thermally persistent
i
CIEEL ) ΦCIEEL
V
(1)
represented by the product of the chemiexcitation yield Φ* (i.e.,
* For convenience, correspondence should be send to Prof. Dr. Waldemar
Adam, Institute of Organic Chemistry, University of Wu¨rzburg, Am
Hubland, D-97074 Wu¨rzburg, Germany. Tel. +49 931 8885340/339.
Telefax: +49 931 8884756. E-mail: adam@chemie.uni-wuerzburg.de.
† Institute of Organic Chemistry, University of Wu¨rzburg.
‡ Tropix, Inc.
(4) Koo, J.-Y.; Schmidt, S. P.; Schuster, G. B. Proc. Natl. Acad. Sci.
U.S.A. 1978, 75, 30-33.
§ Institute of Physical Chemistry, University of Wu¨rzburg.
| Russian Academy of Sciences.
(5) (a) Bronstein, I.; Edwards, B.; Voyta, J. C. J. Biolumin. Chemilumin.
1988, 2, 186. (b) Edwards J. C.; Sparks, A.; Voyta, J. C.; Bronstein, I. J.
Biolumin. Chemilumin. 1990, 5, 1-4. (c) Bronstein, I.; Edwards, B.; Voyta,
J. C. J. Biolumin. Chemilumin. 1989, 4, 99-111.
X Abstract published in AdVance ACS Abstracts, October 15, 1996.
(1) Koo, J.-Y.; Schuster, G. B. J. Am. Chem. Soc. 1977, 99, 6107-6109.
(2) Adam, W.; Cueto, O. J. Am. Chem. Soc. 1979, 101, 6511-6515.
(3) Adam, W.; Zinner, K.; Krebs, A.; Schmalstieg, H. Tetrahedron Lett.
1981, 22, 4567-4570.
(6) (a) Schaap, A. P.; Chen, T.-S.; Handley, R. S.; DeSilva, R.; Giri, B.
P. Tetrahedron Lett. 1987, 28, 1155-1158. (b) Schaap, A. P.; Handley, R.
S.; Giri, B. P. Tetrahedron Lett. 1987, 28, 935-938.
S0002-7863(96)01904-X CCC: $12.00 © 1996 American Chemical Society

Electron Exchange Luminescence of Dioxetanes
J. Am. Chem. Soc., Vol. 118, No. 43, 1996 10401
Kinetic Background and Analysis
Scheme 1
In the case of the CIEEL process shown in Scheme 1, the
phenolate functionality of the dioxetanes 1a (AMPPD) and 1b
(CSPD) is protected by a phosphate group. The CIEEL
cleavage of such dioxetanes may be triggered by alkaline
phosphatase, in which the enzyme removes the phosphate group.
This case constitutes the basic concept in the design of effective
commercial chemiluminescence probes for clinical applica-
tions.5,10,11 The alkaline phosphatase in most applications is
attached to target biomolecules (proteins and nucleic acids) as
a label or is expressed by a specific gene in reporter gene
assays11 and its catalytic action results in the dephosphorylation
of the aryl phosphate moiety of the dioxetane to release the
phenolate functionality (step 1 f 2, Scheme 1), which
subsequently triggers light emission by cleavage of the dioxetane
ring to produce the electronically excited fluorophor (step 2 f
4*, Scheme 1).
Since every enzymatic CIEEL process is pH-controlled, it is
very important to elucidate the pH dependence of the reaction
kinetics. In Scheme 1, the variation of pH may affect (i) the
CIEEL yield through changes of the fluorescence properties of
the CIEEL emitter, (ii) the rate of cleavage of the intermediary
dioxetane phenolate 2, and (iii) the enzymatic dephosphorylation
of the dioxetane 1. The aim of the present study was to assess
the pH effects on these different steps of this important CIEEL
process.
Protonation of excited phenolate 4* may result not only in
the formation of the ground state phenol 4-H but also in excited
phenol 4-H* species (Scheme 1) through adiabatic proton
transfer.12 Thus, apart from the influence on the 4* fluorescence
efficiency, variation of pH may result in a new CIEEL emitter,
namely 4-H*. To verify the possible intervention of such a
new emissive species, merely the pH dependence of the emission
wavelengths needs to be checked.
As to the pH dependence of the fluorescence efficiency for
the phenolate 4*, its measurement presents problems. In buffer
solutions, the ground-state phenolate 4 is in equilibrium with
the phenol 4-H and since the absorption spectra of these two
species are quite overlapped and the relative concentration of
the 4 and 4-H species is also dependent on pH, conventional
measurements of the pH dependence of the steady-state
fluorescence emission are unreliable because the amount of the
emitter derived from 4 varies with pH in the equilibrium 4 h
4-H (Scheme 1). To circumvent this problem, one needs to
conduct time-resolved fluorescence measurements as function
of pH. Since the fluorescence efficiency Φfl is given by Φfl )
kflτ, i.e., the product of the fluorescence rate constant kfl and
the fluorescence lifetime τ, pH changes of the fluorescence
efficiency should be accessible through their effect on the
fluorescence lifetime. Thus, in the present work the measure-
ments of the phenolate 4* fluorescence lifetime Versus pH were
to be performed.
spiroadamantyl-substituted dioxetanes with a protected phenolate
ion. The advantage of such spiroadamantyl-substituted dioxe-
tanes is their thermal persistence and their convenient synthesis
through photooxidation.7 Our detailed kinetic study of the
excited state formation in the thermal decomposition of such
dioxetanes has been reported recently.8
The CIEEL of these dioxetanes can be generated at will on
treatment with an appropriate reagent (trigger) to release the
phenolate ion, which depends on the nature of the protective
group. In early studies of the chemical6 and enzymatic5,6b
triggering, the phenolate moiety (m-oxybenzoate anion) was
found to be the only excited-state decomposition product, hence
the observed CIEEL represents fluorescence of the latter, which
was confirmed by the CIEEL spectra.5c,6
Although the CIEEL phenomenon was intensively studied,
most research efforts were undertaken to elucidate emissive
species and plausible chemiexcitation pathways as well as to
develop efficient CIEEL systems. A comprehensive kinetic
theory of such important and fundamental phenomenon is still
lacking. However, the mechanism and kinetics of the CIEEL
generation need to be studied in detail, if further effective
CIEEL-triggerable systems are to be rationally designed rather
than empirically through trial and error. That is why we have
undertaken model kinetic studies of the phenolate-initiated
intramolecular CIEEL processes. Recently we have reported a
kinetic study of the CIEEL in the decomposition of the silyloxy-
substituted spiroadamantyl dioxetanes triggered by fluoride ions
through the removal of the SiMe2tBu protective group.9 In the
present work we present the kinetic study of the enzymatic
CIEEL process shown in Scheme 1. Through this work we
intend to contribute to the development of the kinetic theory of
the CIEEL phenomenon.
Data on the CIEEL yield at various pH may be available
through the measurements of the total amount of light emitted
in the complete dioxetane decomposition at high alkaline
phosphatase concentration. The total number of photons Nphotons
emitted in the complete dioxetane decomposition is represented
by the area under the CIEEL intensity curve. Since the time
profile of the CIEEL intensity iCIEEL(t) is described by eq 1 and
the area under such a curve is given by integration of iCIEEL(t)
(7) Adam, W.; Arias, L. A.; Zinner, K. Chem. Ber. 1983, 116, 839-
846.
(8) Trofimov, A. V.; Vasil'ev, R. F.; Adam, W.; Mielke, K. Photochem.
Photobiol. 1995, 62, 35-43.
(9) Trofimov, A. V.; Mielke, K.; Vasil'ev, R. F.; Adam, W. Photochem.
Photobiol. 1996, 63, 463-467.
(10) Beck, S.; Ko¨ster, H. Anal. Chem. 1990, 62, 2258-2270.
(11) Bronstein, I.; Fortin, J. J.; Voyta, J. C.; Juo, R.-R.; Edwards, B.;
Olesen, C. E. M.; Lijam, N.; Krichka, L. J. BioTechniques 1996, 17, 172-
177.
(12) Ireland, J. F.; Wyatt, P. A. H. AdV. Phys. Org. Chem. 1976, 12,
131-221.

10402 J. Am. Chem. Soc., Vol. 118, No. 43, 1996
Adam et al.
Scheme 2
Scheme 3
over the reaction time, the expression for Nphotons is given by
eq 2, in which
∞ Vdt
∫
0
represents the total dioxetane concentration [1] decomposed by
the enzyme. Therefore, from eq 2 it follows that the CIEEL
yield ΦCIEEL is determined by eq 3.
synthesis of phosphoric monoesters with alkaline phosphatase.18
In addition to the cleavage k3 and rephosphorylation k-2, in
aqueous buffer the deprotected dioxetane phenolate 2 undergoes
reversible protonation (Schemes 1 and 3). In more detailed
catalytic analyses, a distinction is made14,17 between covalent
binding and noncovalent association of the phosphate and the
enzyme molecule; moreover, the isomerization of the Michaelis
complex EH+‚1 is considered.19 However, such mechanistic
details make the kinetic analysis of the enzymatic CIEEL
process extremely complex. To simplify the kinetic analysis,
we assume the simplified catalytic mechanism in Scheme 3 to
rationalize the experimental results.
The pH effects on the rate-determining step of the catalysis
and on the concentration of the catalytically active enzyme forms
were expected to be reflected in both the catalytic rate constant
(turnover number) kcat and the Michaelis constant KM. For this
reason, these catalytic parameters were to be measured as
function of pH.
For the determination of the catalytic parameters, steady-state
conditions were to be employed in the kinetic analysis. Thus,
when the enzyme concentration e0 is much lower than [1], the
rate-determining step of the overall CIEEL process is repre-
sented by the enzymatic dephosphorylation of the dioxetanes
1. In this case, the reaction rate Vcat is expressed by the
Michaelis-Menten rate law (eq 4). On substitution of eq 4
into eq 1, one obtains eq 6 for the CIEEL intensity at the steady-
state conditions as a function of the dioxetane concentration.
The linear form of eq 5 shown in eq 6 allows to obtain the
catalytic parameters kcat and KM from the experimental depen-
Nphotons
)
∞iCIEEL dt ) ΦCIEEL ∞Vcat dt ) ΦCIEEL[1] (2)
∫
∫
cat
0
0
Nphotons
ΦCIEEL
)
(3)
[1]
The pH dependence of the enzymatic dephosphorylation of
the dioxetanes 1 (step 1 f 2, Scheme 1) derives from the pH
influence on the rate-determining step of catalysis and the
concentration of the catalytically active enzyme form.13 The
pH effect on the latter may consist of direct changes on the
active center of the enzyme (Ser-O-)14 and indirect ones on
the functionalities in the vicinity of the active center. In view
of a complex nature of the pH influence on the active enzyme
form, we simplify our qualitative approach by merely assuming
that the catalytically active form EH+ of the enzyme undergoes
inactivation both through protonation at decreasing and depro-
tonation at the increasing pH. Thus, the catalytically active form
EH+ of the enzyme is in equilibrium with its inactive protonated
(EH22+) and deprotonated (E) forms, as shown in Scheme 2.
The same approach was recently applied to the description of
the pH effect on horseradish peroxidase.15
To understand the pH influence on the catalysis by alkaline
phosphatase at the molecular level, the mechanism of enzyme
action needs to be examined in detail in terms of Michaelis-
Menten kinetics. Although phosphatases from various sources
at the same conditions reveal different catalytic parameters, the
general features of the catalytic mechanism are similar.14 After
the formation of a noncovalent Michaelis complex between the
enzyme and substrate molecule, the catalysis by alkaline
phosphatase proceeds through the phosphoenzyme intermediate
(phosphorylation of the enzyme).16 Release of the free enzyme
molecule from the phosphoenzyme occurs effectively at high
pH, whereas at low pH this process is slow and thereby rate-
determining.17 The pH increase may cause a switchover of
the rate-determining step from dephosphorylation of the enzyme
to its phosphorylation13 by the participation of HO- ions.
Consequently, the simplest catalytic mechanism may be repre-
sented by Scheme 3, in which EH+ is the active enzyme
kcate0[1]
Vcat
)
(4)
[1] + KM
ΦCIEELkcate0[1]
[1] + KM
iCcaItEEL ) ΦCIEELccat
)
(5)
(6)
KM
1
CIEEL -1
(i
)
)
1 +
cat
{
}
ΦCIEELkcate0
[1]
dence of the CIEEL intensity on the dioxetane substrate
concentration [1]. The turnover number kcat may be obtained
from the intercept (ΦCIEELe0 kcat)-1 of the double-reciprocal plot
of this dependence according to eq 6, since e0 is known and
ΦCIEEL may be measured according to eq 3. Division of the
slope of such a plot by its intercept gives the Michaelis constant
KM. These measurements performed at various pH allow one
to obtain pH dependence of the catalytic parameters kcat and
KM. To rationalize kcat and KM Versus the pH experimental data
in terms of Scheme 3, explicit forms of the pH dependence of
these parameters need to be derived.
-
molecule, EH+‚1 is the Michaelis complex, and EH+‚PO3
represents the phosphorylated enzyme. Furthermore, the sub-
strate rephosphorylation step k-2 is also involved in Scheme 3,
since this reaction is known and even used for the enzymatic
(13) Jenks, W. P. Catalysis in Chemistry and Enzymology; McGraw-
Hill, Inc.: New York, 1969; pp 487-490.
(14) Coleman, J. E.; Besman, M. J. A. In Hydrolytic Enzymes; Neuberger,
A., Brocklehurst, K., Eds.; Elsevier Science Publishers B. V.: Amsterdam-
New York-Oxford, 1987; pp 337-406.
(15) Holzbaur, I. E.; English, A. M.; Ismail, A. A. J. Am. Chem. Soc.
1996, 118, 3354-3359.
(18) Pradines, A.; Klaebe, A.; Perie, J.; Paul, F.; Monsan, P. Tetrahedron
1988, 44, 6373-6386.
(19) Gutfreund, H. Enzymes: Physical Principles; Wiley-Interscience:
London-New York, 1972; pp 194-202.
(16) Engstro¨m, L. Biochem. Biophys. Acta 1961, 52, 49-59.
(17) Chappelet-Tordo, D.; Fosset, M.; Iwatsubo, M.; Gache, C.; Laz-
dunski, M. Biochemistry 1974, 13, 1788-1795.

Electron Exchange Luminescence of Dioxetanes
J. Am. Chem. Soc., Vol. 118, No. 43, 1996 10403
Figure 1. Fluorescence decay curves (λex ) 337 nm) and weighted residual plots of phenolate 4 ([4] + [4-H] ) 10-4 M) in 0.05 M carbonate
buffer, 1 mM MgCl2, in the presence of 10-8 M alkaline phosphatase at (a) pH 8.68 (τ ) 9.6 ns) and (b) pH 10.3 (τ ) 10.3 ns) at room temperature
(ca. 20 °C).
To permit an experimental distinction between the pH effects
on the catalytic generation and the cleavage of the CIEEL-active
dioxetane 2, the kinetics of the base-induced CIEEL triggering
of its protonated form 2-H Versus pH needed to be studied
independently. The desired rate constant k3 of the dioxetane 2
decomposition (Scheme 3) was to be obtained from the pseudo-
first-order kinetics of the dioxetane 2-H decay at high pH.
Indeed, high pH causes rapid deprotonation of 2-H, followed
by the first-order CIEEL decay of 2. The kinetics is described
CIEEL
by eq 7, in which i2
represents the CIEEL intensity of 2,
and in logarithmic form it is expressed by eq 8. The rate
constant k3 is given by the slope of the plot of ln(i2CIEEL) Versus
reaction time according to eq 8.
iC2 IEEL ) ΦCIEELk3[2] ) ΦCIEELk3[2-H]0e-k t
ln(i2CIEEL) ) ln(ΦCIEELk3[2-H]0) - k3t
(7)
(8)
3
Figure 2. Fluorescence lifetime of the phenolate 4 ([4] + [4] ) 10-4
M) Versus pH in 0.05 M carbonate buffer, 1 mM MgCl2 and in the
presence of 10-8 M alkaline phosphatase at room temperature (ca. 20
°C).
Results
Cleavage Kinetics of the Dioxetane Phenolate Anion. At
pH > 12.6, the CIEEL decay was independent of pH in the
base-induced triggering of dioxetane 2a-H, and its kinetics
followed the pseudo-first-order rate law for the cleavage of the
dioxetane anion 2a (Figure 5). From the linear plot of Figure
5, the rate constant k3 of the dioxetane 2a cleavage was found
to be (1.2 ( 0.04) × 10-2 s-1 at 37 °C according to eq 8.
Fluorescence Lifetime of the CIEEL Emitter Wersus pH.
The fluorescence decay curves at the lowest and highest pH
used are shown in Figure 1. The decay curves at every pH
were well fitted by monoexponential kinetics, which implies
only one emissive species. This was also confirmed by the
coincidence of the normalized emission spectra at the same pH
values. The least-squares iterative deconvolution procedure
applied to all of the decay curves afforded the fluorescence
lifetimes of the phenolate 4 as a function of pH (Figure 2). All
lifetime data in Figure 2 are the average of five measurements
with low values of ø2 (<1.1). Thus, at alkaline pH the
fluorescence lifetime (ca. 10 ns) of the phenolate 4 is indepen-
dent of pH (Figure 2).
CIEEL Yields Wersus pH. According to eq 3, the CIEEL
yields were obtained through measurements of the total amount
of light (areas under the CIEEL curves calibrated against the
Hastings-Weber scintillation “cocktail” 20) emitted in the com-
plete decomposition of the AMPPD and CSPD at high alkaline
phosphatase concentration (10-8 M). CIEEL curves at various
pH are shown in Figure 3. Although at various pH the CIEEL
reveals different kinetics (Figure 3), the areas under the CIEEL
curves and, therefore, the CIEEL yields were found to be
independent of pH within the experimental error (Figure 4). The
values of the CIEEL yields are (7.5 ( 0.3) × 10-6 for the
AMPPD and (5.7 ( 0.3) × 10-6 for the CSPD.
pH Dependence of the Michaelis Constant. The experi-
mental dependences of the stationary CIEEL intensity on the
AMPPD and CSPD concentrations measured at various pH are
shown in Figures 6a and 7a. From the double-reciprocal plots
according to eq 6 (Figures 6b and 7b), the value of the Michaelis
constant was obtained for every pH value used. The experi-
mental pH dependence of the KM is shown in Figure 8, every
point of which represents the average KM value of four
independent measurements for every particular pH.
Catalytic Rate Constant (Turnover Number). At pH 9.0-
10.5 the intercepts of the double-reciprocal plots according to
eq 6 (Figures 6b and 7b) and, consequently, the values of
Φ
CIEELe0kcat are independent of pH for both AMPPD and CSPD.
The latter values were found to be (1.9 ( 0.3) × 10-14 Ms-1
for AMPPD and (1.22 ( 0.21) × 10-14 Ms-1 for CSPD at
4.7 × 10-13 M alkaline phosphatase and 37 °C. On substitution
of the measured CIEEL yields ΦCIEEL for both AMPPD and
CSPD, the turnover number kcat for the enzymatic dephospho-
rylation of the AMPPD was found to be (5.4 ( 0.9) × 103 s-1
and (4.6 ( 0.8) × 103 s-1 for CSPD at pH 9.0-10.5 and 37
°C.
(20) Hastings, J. W.; Weber, G. J. Opt. Am. Soc. 1963, 53, 1410-1415.

10404 J. Am. Chem. Soc., Vol. 118, No. 43, 1996
Adam et al.
Figure 3. pH Dependence of the CIEEL kinetics in the decomposition of (a) AMPPD ([AMPPD] ) 1.65 × 10-5 M) and (b) CSPD ([CSPD] )
1.85 × 10-5 M) catalyzed by alkaline phosphatase ([AP] ) 10-8 M) in 0.05 M carbonate buffer, 1 mM MgCl2 at 37 °C.
Scheme 1, namely (i) phenolate 4 fluorescence, (ii) dioxetane
anion 2 cleavage, and (iii) enzymatic catalysis at alkaline pH is
essential.
In general, according to Scheme 1, protonation of the excited
phenolate 4* may form both the ground (4-H) and the excited
state phenol species (4-H*). The latter adiabatic proton
transfer12 would result in the new CIEEL emitter 4-H* (Scheme
1). However, time-resolved fluorescence measurements showed
that the role of the protonation of 4* is negligible at alkaline
pH since no difference in the phenolate 4* fluorescence lifetimes
at various alkaline pH was observed (Figure 2). This lack of
pH dependence of the phenolate 4 fluorescence lifetime τ can
be justified through a conventional Stern-Volmer analysis at
alkaline conditions.
Let us consider protonation of the excited phenolate 4* as
Figure 4. pH Dependence of the CIEEL yields in the alkaline-
its quenching by H+ with the overall quenching rate constant
phosphatase-catalyzed decomposition of AMPPD (b) and CSPD (9)
kq, irrespective of whether such process results in the ground
at the experimental conditions of Figure 3.
or excited state phenol species 4-H and 4-H*. In the presence
of H+, the conventional Stern-Volmer expression for the
fluorescence lifetime is given by τ ) τ0/(1 + kqτ0[H+]), where
τ0 represents the lifetime of 4* at negligible [H+]. Comparison
of τ and τ0 allows us to assess how significant the protonation
of 4* is at a given pH by estimating τ0 from the experimental
data available for τ. The upper limit of the quenching effect of
the phenolate 4* through its protonation would require the rate
constant kq of this process to be a diffusion-controlled (ca. 1010
M-1 s-1), which means that at pH 9, for example, and the
experimental value τ ca. 10 ns (Figure 2), τ ) τ0/(1 + 10τ0) ≈
10-8 s. Therefore, τ0 ≈ 10-8/(1-10-7) ≈ 10-8 s, i.e., there is
no difference in the lifetime values at pH 9 and in the absence
of protons. This implies that in the applied pH range 9.0-
10.5, protonation of the excited phenolate 4* is negligible
because of its short lifetime. Hence, under alkaline conditions,
Figure 5. CIEEL decay of the 2a-H ([2a-H]0 ) 2.42 × 10-5 M)
neither the nature of the CIEEL emitter phenolate 4* nor its
fluorescence lifetime and, therefore, its fluorescence efficiency
are affected by pH.
decomposition induced by NaOH in water (pH 12.7) at 37 °C; first-
order plot according to eq 8 (a) of the CIEEL intensity Versus time
data (b).
The chemiexcitation step (2 f 4* in Scheme 1) consists of
the elementary act of the cleavage of the dioxetane 2 and is
expectedly independent of pH. Therefore, pH may affect the
CIEEL yield only by changes of the fluorescence efficiency of
the CIEEL emitter. However, we have just shown that pH does
not affect the fluorescence efficiency and, therefore, pH should
not affect the CIEEL yield as well as experimentally established
in Figure 4.
Thus, under alkaline conditions, the fate of the CIEEL emitter
is independent of pH because of its short lifetime (τ ca. 10
ns). On the contrary, its precursor, the dioxetane phenolate 2
is a long-lived species. Indeed, from the pseudo-first-order
Discussion
All kinetic parameters for the enzymatic CIEEL process of
Scheme 1, namely the CIEEL emitter fluorescence lifetime, the
rate constant of the dioxetane anion 2 cleavage, the Michaelis
constant, and the turnover number as well as the CIEEL
efficiency were measured within the pH range at which alkaline
phosphatase is catalytically active. At pH > 9, the Michaelis
constant KM depends on pH, whereas all the other parameters
are independent of pH. To rationalize these experimental
results, a detailed discussion of the interrelated processes in

Electron Exchange Luminescence of Dioxetanes
J. Am. Chem. Soc., Vol. 118, No. 43, 1996 10405
Figure 6. CIEEL in the decomposition of AMPPD catalyzed by alkaline phosphatase ([AP] ) 4.7 × 10-13 M) in 0.05 M carbonate buffer, 1 mM
MgCl2 at 37 °C and at various pH: (a) intensity Versus AMPPD concentration and (b) double-reciprocal plot according to eq 6.
Figure 7. CIEEL in the decomposition of CSPD catalyzed by alkaline phosphatase ([AP] ) 4.7 × 10-13 M) in 0.05 M carbonate buffer, 1 mM
MgCl2 at 37 °C and at various pH; (a) intensity Versus CSPD concentration and (b) double-reciprocal plot according to eq 6.
CIEEL decay at high pH (Figure 5), the rate constant k3 of the
dioxetane 2a cleavage is (1.2 ( 0.04) × 10-2 s-1 at 37 °C.,
which corresponds to a half-life (τ1/2 ) ln 2/k3) of ca.1 min.
This means that participation of 2 in bimolecular processes such
as its rephosphorylation and protonation (Scheme 3) may be
very probable. These possibilities need now to be scrutinized.
Unfortunately, involvement of the rephosphorylation step k-2
in Scheme 3 makes for very complex kinetics. However,
knowledge of the rate constant k3 should allow us to decide
whether step k-2 in Scheme 3 can be neglected and thereby
phosphorylation of the enzyme considered to be irreversible.
Such a simplification makes the kinetic analysis of the enzymatic
triggering process amenable. Let us compare the rates of the
two competitive steps k3 Versus k-2, which are the cleavage of
the dioxetane 2 and its rephosphorylation. If, according to
Scheme 2, the three forms EH22+, EH+, and E of the free
enzyme are considered, of which only form EH+ is catalytically
active, the rate of rephosphorylation is given by the expression
k-2[2][EH+‚PO3-], while the cleavage rate of 2 is k3[2]. Since
[2] in both rate expressions is the same, one needs to compare
the values for k-2[EH+‚PO3-] and k3 (ca.10-2 s-1). The upper
limit for the value k-2[EH+‚PO3-] would require k-2 to be
diffusion-controlled (ca.1010 M-1 s-1) and [EH+‚PO3-] to be
equal to the initial, total enzyme concentration e0 (ca. 10-13
M), which would amount to ca. 10-3 s-1. Thus, even the upper
limit of k-2[EH+‚PO3-] is by about an order of magnitude
smaller than k3. The real k-2[EH+‚PO3-] value should be much
less than 10-3 s-1, since very probably k-2 is less than diffusion-
controlled and [EH+‚PO3-] should be much lower than the total
enzyme concentration e0. For the latter, not all of the enzyme
Scheme 4
molecules are catalytically active and, moreover, not all of
catalytically active enzyme molecules are phosphorylated, i.e.,
[EH+‚PO3-] < [EH+ ] < e0 (ca. 10-13 M). Consequently, for
the intermediate 2a, it follows that k-2[EH+‚PO3-] , k3, while
for the chloro-substituted dioxetane intermediate 2b, this
inequality is even more pronounced. Indeed, it was previously
found9 that chloro substitution leads to faster cleavage of 2 in
every medium studied. Therefore, one can neglect the rate of
the rephosphorylation (k-2) in comparison with the rate of the
dioxetane 2 cleavage (k3) and thereby consider phosphorylation
of the enzyme (k2) in the Michaelis complex (Scheme 3) as an
irreversible step, which considerably simplifies the kinetics of
the enzymatic triggering process.
The influence of protonation of the intermediate 2 on its
stationary concentration and thereby CIEEL intensity may be
understood through the following kinetic analysis. According
to Scheme 4, the pseudo-constants k˜5 of the protonation of the
dioxetane phenolate 2 and k˜5 of the deprotonation of the phenol
2-H in aqueous buffer may be defined by eqs 9 and 10. Neglect
of the rephosphorylation step k-2 in Scheme 3 gives eqs 11
and 12 for the concentrations of 2 and 2-H under the steady-
state conditions. Solution of eqs 11 and 12 in terms of [2]
results in eq 13, from which one can see that under these circum-

10406 J. Am. Chem. Soc., Vol. 118, No. 43, 1996
Adam et al.
k˜5 ) k5[H2O] + k′[H+]
(9)
In the presence of the dioxetane 1 as substrate, the Michaelis
5
-
complex EH+‚1 and the phosphorylated enzyme EH+‚PO3
k˜-5 ) k-5[HO-] + k′ [H2O]
(10)
need to be considered in addition to the free enzyme forms.
According to Scheme 3, and neglect of the dioxetane rephos-
phorylation step k-2, the concentrations of EH+‚1 and EH+‚PO3-
under the steady-state conditions are expressed by eqs 18 and
19. From eq 18 one obtains eq 20, which relates [EH+] with
[EH+‚1], and eq 19 gives similarly eq 21 for [EH+‚PO3-] and
[EH+‚1].
-5
d[2]
) Vcat + k˜-5[2-H] - (k˜5 + k3)[2] ) 0
dt
(11)
d[2-H]
) k˜5[2] - k˜-5[2-H] ) 0
dt
(12)
(13)
Vcat ) k3[2]
d[EH+‚1]
) k1[EH+][1] - (k-1 + k2)[EH+‚1] ) 0 (18)
dt
stances, the concentration of the dioxetane phenolate 2 is equal
to Vcat/k3. Thus, under the steady-state conditions, the proto-
nation of 2 and the deprotonation of 2-H do not affect the dioxe-
tane 2 concentration and thereby the stationary CIEEL intensity.
To understand how enzymatic dephosphorylation of the
dioxetanes AMPPD and CSPD depends on pH, the experi-
mental data on the catalytic parameters kcat and KM Versus pH
need to be examined in terms of Scheme 3. The kcat value of
ca. 5 × 103 s-1 shows rapid turnover of the alkaline phosphatase
for both AMPPD and CSPD. As to the question whether kcat
is pH dependent, we had shown that the value ΦCIEELe0kcat is
independent of pH within the entire pH range (9.0-10.5) at
which the alkaline phosphatase catalyzes effectively. Indeed,
the intercept (ΦCIEELe0kcat)-1 of the double-reciprocal plot
according to eq 6 (Figures 6b and 7b) was found to be
independent of pH within the pH range of 9.0-10.5. Since
ΦCIEEL is also independent of pH, it follows that kcat is
independent as well. Thus, for the entire pH range employed
here, kcat is ca. 5 × 103 s-1. Only at pH lower than 9, the
intercept (ΦCIEELe0kcat)-1 of the double-reciprocal plot (Figure
6b) increases markedly with decreasing pH, which reflects the
decrease of the kcat since ΦCIEEL and e0 are constant values.
Similar pH dependence of the kcat is known for the hydrolysis
of p-nitrophenylphosphate by intestinal alkaline phosphatase.17
In contrast to the turnover number (kcat), the Michaelis
constant (KM) depends on pH within the entire pH range used.
To understand this pH behavior of the catalytic parameters kcat
and KM, one must take into account the existence of various
forms of the free enzyme. According to Scheme 2, the
concentrations of the three forms EH22+, EH+, and E of the
free enzyme can be related with one another by the ionization
constants15,21 Ka and Ka defined by eqs 14 and 15. This leads
-
d[EH+‚PO3 ]
-
) k2[EH+‚1] - k4[HO-][EH+‚PO3 ] ) 0
dt
(19)
(k-1 + k2)[EH+‚1]
[EH+] )
(20)
(21)
-
k1[1]
k2
-
[EH+‚PO3 ] )
[EH+‚1]
k4[HO-]
[H+]
Ka
2
e0 ) [EH+] 1 +
+
+ [EH+‚1] + [EH+‚PO3 ]
+
{
}
K
a1
[H ]
(22)
In the presence of the substrate 1, the total enzyme concentra-
tion e0 consists of the sum of the concentrations of all its forms,
namely EH22+, EH+, E, EH+‚1, and EH+‚PO3-, which is
expressed in eq 22. Solution of eqs 20-22 results in eq 23 for
the concentration of the Michaelis complex. The rate of the
[EH+‚1] )
e0[1]
[H+]
Ka
k2
k-1 + k2
1
1 +
[1] +
1 +
+
+
-
+
(
)
)
(
)
k2
K
k4[HO ]
a1
[H ]
k1 1 +
-
{
}
(
)
)
k4[HO ]
(23)
Vcat
)
k2e0[1]
1
2
to eq 16 for the total enzyme concentration e0 as a sum of the
concentrations of all free enzyme forms, from which it follows
that the concentration of the catalytically active enzyme form
EH+ in the absence of substrate is given by eq 17.
[H+]
Ka
k2
k-1 + k2
2
1 +
[1] +
1 +
-
+
(
(
)
k2
K
a1
k4[HO ]
[H ]
k1 1 +
-
{
}
(
k4[HO ]
(24)
[EH+][H+]
[EH22+]
Ka )
(14)
(15)
catalytic generation of the CIEEL-active dioxetane 2 through
enzymatic dephosphorylation (Schemes 3) can be obtained from
eq 23 by multiplication of the latter by the rate constant k2,
which leads to eq 24. Comparison of eq 24 with the Michaelis-
Menten eq 4 leads to expressions for the catalytic parameters
kcat and KM in the form of eqs 25 and 26, which account for the
experimentally observed pH influence.
1
[E][H+]
[EH+]
Ka )
2
[H+]
Ka
2
e0 ) [EH22+] + [EH+] + [E] ) [EH+] 1 +
+
+
{
}
K
a1
[H ]
k2
(16)
(17)
kcat
)
(25)
k2
e0
[H+]
1 +
[EH+] )
k4[HO-]
Ka
2
[H+]
1 +
+
Ka
k-1 + k2
a1
[H+]
2
K
KM )
1 +
+
(26)
+
{
}
k2
K
a1
[H ]
k1 1 +
(21) Mathews, C. K.; van Holde, K. E. Biochemistry; The Benjamin/
Cummings Publishing Co: Redword City, 1990; pp 43-137.
-
(
)
k4[HO ]

Electron Exchange Luminescence of Dioxetanes
J. Am. Chem. Soc., Vol. 118, No. 43, 1996 10407
term contributes mainly to KM in eq 28 since it is this term
which is responsible for the increase of the KM (Figure 8) with
increasing pH (decreasing [H+]). In terms of Scheme 2, the
latter fact is presumably associated with the decrease of the
concentration of the active enzyme form EH+ through its
deprotonation at increasing pH.
From the above detailed kinetic analysis of the experimental
data on the enzymatic triggering of dioxetanes 1 we observe
that although every step of the triggering process in Schemes 1
and 3 involves either H+ or HO- ions, at alkaline pH and steady-
state conditions, only the enzymatic dephosphorylation of the
AMPPD and CSPD depends on pH in this CIEEL process. The
turnover number (kcat) is affected only at pH below 9, which
reflects a switchover of the rate-determining step of the enzyme
dephosphorylation to its phosphorylation (Scheme 3) at increas-
ing pH and is described by eq 25 and its limiting form of eq
27. The Michaelis constant (KM) depends on pH within the
entire pH range used, which may be attributed to the pH effect
on the concentration of the catalytically active enzyme form.
We conclude that the protonation of 2 and the deprotonation
of 2-H do not affect the CIEEL intensity under steady-state
conditions and the CIEEL efficiency is independent of pH.
Fortunately, the neglect of the dioxetane rephosphorylation step
k-2 (Scheme 3) at alkaline pH simplifies the kinetics of the
CIEEL process sufficiently to permit a complete analysis. Such
kinetic analysis provides the essential mechanistic insight for
the design of novel enzyme-triggered CIEEL-active probes.
Particularly when target molecules (proteins or nucleic acids)
are attached to the enzyme, the catalytic properties of the latter
are expected to be affected through the pH dependence of both
the turnover number (kcat) and the Michaelis constant (KM)
compared with those of the free enzyme. Therefore, the pH
conditions should be optimized for every particular enzyme-
triggered chemiluminescent probe through a detailed kinetic
analysis, especially when enzymes immobilized on membranes
and water-soluble polymers are employed.
Figure 8. pH Dependence of the Michaelis constant for the alkaline-
phosphatase-catalyzed decomposition of AMPPD (b) and CSPD (9)
in 0.05 M carbonate buffer at 37 °C, 1 mM MgCl2 and 4.7 × 10-13
M
alkaline phosphatase.
Indeed, the lack of pH dependence of the kcat at pH > 9
suggests that at high pH the rate-limiting step is the phospho-
rylation of the enzyme, whereas the regeneration of the free
catalytically active enzyme form EH+ through the pH-controlled
dephosphorylation of EH+‚PO3- is fast, since k4[HO-] is high
enough. This reduces eq 25 to its limiting case shown in eq
27, which is valid at pH > 9, i.e., high [HO-]. Similarly, at
pH > 9, eq 26 for the Michaelis constant can be reduced to eq
28 as limiting case, since again k4[HO-] is sufficiently high.
kcat ≈ k2
k-1 + k2
(27)
[H+]
K
a2
KM ≈
1 +
+
(28)
+
{
}
k1
K
a1
[H ]
The pH dependence of the Michaelis constant KM is thereby
determined by the relative importance of the terms
[H+]
Acknowledgment. Generous funding by the Deutsche For-
schungsgemeinschaft (Sonderforschungsbereich 172 “Moleku-
lare Mechanismen kanzerogener Prima¨rvera¨nderungen”) is
gratefully appreciated. A.V.T. and R.F.V. thank also the
Rossiiskii Fond Fundamental'nykh Issledovanii (Grant No. 96-
03-34142) and the International Science Foundation (Grant JIL
100) for the financial support. We thank Dr. C. R. Saha-Mo¨ller,
Dr. J. C. Voyta, and K. Mielke for many helpful discussions
and comments.
Ka
1
and
Ka
2
[H+]
in eq 28. The shape of the experimental pH dependence of the
KM (Figure 8) suggests that within the pH range used, the
Supporting Information Available: Experimental proce-
dures (2 pages). See any current masthead page for ordering
and Internet access instructions.
Ka
2
[H+]
JA961904G