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.