Design and synthesis of chemiluminescent probes for the detection of cholinesterase activity

Article abstract of DOI:10.1021/ja0171299

Acetylcholinesterase is one of the most widely used and studied enzymes. Not only does this enzyme regulate neurotransmission (and thus play a key role in neurodegenerative processes) but it is also a prime target for pest control agents and warfare agent

Full text of DOI:10.1021/ja0171299

Published on Web 04/05/2002
Design and Synthesis of Chemiluminescent Probes for the
Detection of Cholinesterase Activity
Ste´phane Sabelle,† Pierre-Yves Renard,*,‡ Karine Pecorella,§
Sophie de Suzzoni-De´zard,‡ Christophe Cre´minon,§ Jacques Grassi,*,§ and
Charles Mioskowski‡,|
Contribution from SPI Bio, 2, rue du Buisson aux Fraises, ZI de la Bonde 91741 Massy Cedex,
France, CEA, SerVice des Mole´cules Marque´es, DBCM/DSV, CE Saclay 91191 Gif sur YVette
Cedex, France, CEA, SerVice de Pharmacologie et d’Immunologie, DRM/DSV, CE Saclay 91191
Gif sur YVette Cedex, France, and Laboratoire de Synthe`se Bioorganique (UMR 7514), 74,
route du Rhin 67401 Illkirch-Graffenstaden, France
Received September 21, 2001
Abstract: Acetylcholinesterase is one of the most widely used and studied enzymes. Not only does this
enzyme regulate neurotransmission (and thus play a key role in neurodegenerative processes) but it is
also a prime target for pest control agents and warfare agents. Above all, due to its particularly high turnover
rate, acetylcholinesterase is among the most efficient reporter enzymes yet described (for use as enzymatic
tracer in immunoassays, for instance). However, its activity is detected through a colorimetric reagent, the
Ellman reagent, which displays low detection limits and is often subject to background perturbations. In
the course of our search for a more sensitive detection assay, we describe here a first-generation 1,2-
dioxetane chemiluminescent probe, based on chemically induced electron exchange luminescence, which
has an approximately 10 times lower detection limit than the Ellman colorimetric assay (2.5 × 10-19 mol
for Electrophorus electricus AChE in its tetrameric form).
Scheme 1. Principle of AChE Detection with the Ellman Reagent
Introduction
Acetylcholinesterase (AChE, EC 3.1.1.7) is a well-known and
thoroughly studied enzyme involved in the regulation of the
concentration of neurotransmitter acetylcholine at cholinergic
synapses.1 Its particularly high catalytic potency makes it the
ultimate candidate for use as an enzymatic tracer2 (turnover,
64 000 s-1, three times the turnover of horseradish peroxidase).
However, since the first description of the Ellman reagent in
the late 1950s, no analytical breakthrough for the detection of
its catalytic activity has been described. The Ellman reagent is
a buffered mixture of two products. First, acetylthiocholine
iodide, a pseudosubstrate of AChE, whose rate of hydrolysis
almost reaches that of acetylcholine itself. Second, 5,5′-dithio
2-nitrobenzoic acid, a chromogenic reagent. Once acetylthio-
choline iodide has been hydrolyzed into thiocholine iodide and
acetic acid, the liberated nucleophilic thiol adds to the disulfide,
yielding disulfide 1 and yellow 5-mercapto 2-nitrobenzoic acid
2 (ꢀ ) 13 600 at 414 nm) (Scheme 1). Through colorimetric
detection, the amount of detectable Electrophorus electricus
AChE (in its tetrameric form) has been estimated to 1.85 ×
10-18 mol.3
In the course of our search for a more sensitive detection
assay, which could be used both in 96- and 384-well microtiter
plates for immunoassays and for histochemical or immunohis-
tochemical analyses, we focused our attention on a chemilu-
minescent reagent for the detection of AChE activity. The
coupling of enzymatic reactions to chemiluminescence (or
† SPI Bio.
‡ CEA, Service des Mole´cules Marque´es.
§ CEA, Service de Pharmacologie et d’Immunologie.
| Laboratoire de Synthe`se Bioorganique.
(1) Massoulie´, J.; Pezzementi, L.; Bon, S.; Krejci, E.; Vallette, F. M. Prog.
Neurobiol. 1993, 41, 31-91.
(2) (a) Antoine, C.; Lellouche, J.-P.; Maclouf, J.;. Pradelles, P. Biochim.
Biophys. Acta 1991, 1075, 162-168. (b) Pradelles, P.; Grassi, J.; Maclouf,
J. Anal. Chem. 1985, 57, 1170-1173.
(3) Grassi, J. Ph.D. Dissertation, Paris VI University, Paris, France, 1990.
9
4874
J. AM. CHEM. SOC. 2002, 124, 4874-4880
10.1021/ja0171299 CCC: $22.00 © 2002 American Chemical Society

Chemiluminescent Probes for Cholinesterase Activity
A R T I C L E S
Scheme 2. Structures of the Chemiluminescent Probes
Scheme 3. Structure of the Thiol Detection Chemiluminescent
Probe
bioluminescence) phenomena allows detection of enzymes (or
substrates, or inhibition) at very low concentrations.4
Three main chemiluminescent reactions have been used in
bioanalytical chemistry:5 oxidation of luminol catalyzed by
enzymes (HRP, horseradish peroxidase, for instance) or metal
complexes; peroxyoxalate chemiluminescent reaction;5d,6 and
1,2-dioxetane or 1,2-dioxetanone ring opening through CIEEL
(chemically induced electron exchange luminescence),5c,7 whose
exact mechanism is still the subject of many studies.8 Luminol
oxidation has already been used in two complex multistep
multienzymatic reactions to detect AChE activity.9,10 In our
hands, and despite a few technical improvements,11 these com-
plicated multistep procedures proved unsuitable mainly on two
grounds. First, they are very sensitive to enzymatic, metallic,
or chemical contaminants. Second, sensitive detection is only
possible in a two-step detection: first production of hydrogen
peroxide, a relatively unstable and particularly reactive species,
then detection of the H2O2 produced. Superposition of these
two steps dramatically lowers the detection sensitivity.11 Kinetic
analysis is then hardly feasible, and only end-point measure-
ments are of practical use.
By analogy with the chemiluminescent 1,2-dioxetane sub-
strates 3 used for the detection of alkaline phosphatase,12 we
decided to evaluate this CIEEL with enzymatically triggered
hydrolysis of 1,2-dioxetane ester 4 by AChE (Scheme 2). Use
of this kind of substrate has already been described for the
detection of the catalytic activity of carboxylesterases.13 Un-
fortunately, the adamantyl moiety, required for the stability of
the dioxetane moiety of 4, proved too bulky to allow 4 to reach
the AChE catalytic site, buried deep in a narrow hydrophobic
gorge.14
We thus designed an indirect chemiluminescent disulfide
probe, TDP, as depicted in Scheme 3. The structure of this
reagent is based on the same reaction cascade as for the Ellman
reagent. AChE hydrolysis of acetylthiocholine iodide leads to
thiocholine iodide. Its nucleophilic thiol moiety should then
break the disulfide bond of the chemiluminescent probe to yield
an aromatic thiophenolate, whose structure is closely related to
that of the phenolate produced by hydrolysis of phosphate 3.
Through cleavage of the 1,2-dioxetane ring, CIEEL should then
lead to an excited m-mercaptobenzoate anion, whose decay to
the ground state should provide light, as for the corresponding
m-oxobenzoate anion.
Besides the disulfide bond, the structure of the chemilumi-
nescent reagent should be divided into four parts:
(1) the 1,2 dioxetane ring, whose decay will give rise to
luminescence;
(2) an adamantyl moiety to stabilize the 1,2-dioxetane ring
[if solubility problems are encountered, an additive charged
group (Z ) CO2-, SO3-) or halogen atom (Z ) Cl) could be
introduced on the adamantyl ring];
(3) an aromatic electron-transferring moiety, which, once the
disulfide bond is broken and the thiophenolate formed, will
trigger the 1,2-dioxetane ring opening, via electron transfer as
in phenolate 3; and
(4) an orienting aromatic moiety, substituted with electron-
withdrawing groups (EWG), to remove electrons from its
directly attached sulfur atom, and so center the nucleophilic
addition of thiocholine iodide on this sulfur atom, as depicted
in Scheme 4.
(4) Mayer, A.; Neuenhofer, S. Angew. Chem., Int. Ed. Engl. 1994, 33, 1044-
1072.
(5) (a) Roda, A.; Pasini, P.; Guardigli, M.; Baraldini, M.; Musiani, M.; Mirasoli,
M. Fresenius’ J. Anal. Chem. 2000, 366(6-7), 752-759. (b) for a general
review on chemiluminescence mechanisms, see: Mc Capra, F. M. Methods
Enzymol. 1999, 305, 1-47, and the whole vol. 305 for applications in
enzymology. (c) Schuster, G. B. Acc. Chem. Res. 1979, 12, 366. (d) Garcia-
Campana, A. M.; Baeyens, W. R. G. Analysis 2000, 28, 686-698.
(6) (a) Givens, R. S.; Schowen, R. L. Chemiluminescence and photochemical
reaction detection in chromatography; Birks, J., Ed; VCH: Weinheim,
1989; pp 125-147. (b) Orosz, G.; Givens, R. S.; Schowen, R. L. Crit.
ReV. Anal. Chem. 1996, 26, 1-27.
(7) Dixon, B. G.; Schuster, G. B. J. Am. Chem. Soc. 1981, 103, 3068.
(8) Adam, W.; Matsumoto, M. Trofimov, A. V. J. Org. Chem. 2000, 65, 2078-
2082 and references therein.
Results and Discussion
(9) (a) Israel, M.; Lesbats, B. J. Neurochem. 1981, 37, 1475-1483. (b) Taylor,
S. J.; Haggbald, J.; Greenfield, S. A. Neurochem. Int. 1989, 15, 199-205.
(10) Arakawa, H.; Mae da, M.; Tsuji, A. Anal. Biochem. 1991, 199, 238-242.
(11) Pecorella, K. Ph.D. Dissertation, Paris V University, Paris, France, 1995.
(12) (a)Bronstein, I.; Edwards, B.; Voyta, J. C. J. Biolumin. Chemilumin. 1988,
2, 186. (b) Bronstein, I.; Voyta, J. C.; Thorpe, G. H. G.; Kricka, L. J.;
Amstrong, G. Clin. Chem. 1989, 35, 1441-1146. (c) Edwards, B.; Sparks,
A.; Voyta, J. C.; Strong, R.; Murphy, O.; Bronstein, I. J. Org. Chem. 1990,
55, 6225-6229.
To evaluate this strategy, two simple disulfide reagents, 5
and 6, were first synthesized. The retrosynthetic scheme for these
(13) Schaap, P.; Handley, R. S.; Giri, B. P. Tetrahedron Lett. 1987, 28, 935-
938
(14) Harel, M.; Schalk, I.; Ehret-Sabatier, L.; Bouet, F.; Goeldner, M.; Hirth,
C.; Axelsen, P.; Silman, I.; Sussman, J. Proc. Natl. Acad. Sci. U.S.A. 1993,
90, 9031.
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Scheme 4. Proposed Mechanism for Light Emission with the Designed Chemiluminescent Reagents
Scheme 5. Retrosynthetic Scheme for Chemiluminescent
Reagents 5 and 6
Scheme 6. Formation of Ethyl 3-(S-tert-butylsulfanyl)benzoate 9a
a (i) KI (15 equiv), CuI (5 equiv), HPMT, 155 °C, 3 days; (ii) Bu3SnCl,
Et3N, CCl4, 18 h; (iii) Pd(PPh3)4, toluene, 110 °C, 48 h.
Scheme 7. McMurry Coupling on 9a
two reagents is based on two observations. First, the 1,2-
dioxetane ring is extremely sensitive and has to be introduced
at the very last step of the synthesis, through mild and carefully
controlled oxidation, to prevent co-oxidation of the sulfur atoms.
Second, the adamantyl enol ether is usually introduced via
intermolecular McMurry condensation of adamantanone on the
corresponding ethylbenzoate.12,13 McMurry reaction conditions
are harshly reducing and incompatible with a disulfide bond.
The following retrosynthetic scheme (Scheme 5) was designed
for chemiluminescent probes 5 and 6.
After unfruitful attempts to perform the McMurry coupling
with unprotected or conventional S-protected thiophenols (as
methyl or benzyl thioether, acetic or benzoic thioester), we chose
to introduce a tert-butyl group on the thiophenol moiety. Ethyl
3-(S-tert-butylsulfanyl)benzoate 9 was formed in fairly good
yields through a Stille-like coupling15 of tributyltin tert-bu-
tylsulfanyl 8 on the aromatic iodide 7 (obtained by transhalo-
genation of commercially available ethyl 3-bromobenzoate)16
(Scheme 6).
a (i) TiCl3 (10 equiv), LiAlH4 (5equiv), Et3N (6 equiv), adamantanone,
THF, 70 °C, 20 h.
activated sulfenyl chlorides on protected cysteine.17 Yet coupling
of 2-nitrophenyl sulfenyl chloride on S-tert-butylsulfanyl 10
using acetic acid as solvent, as described, ended in complete
hydrolysis of the enol ether moiety. We thus turned to milder
reaction conditions and carefully monitored the reaction. We
found that when the substitution was performed in THF and
when the course of the reaction was stopped after ca. one-third
of the enol ether 10 was consumed, an acceptable yield of 26%
of disulfide 1 could be isolated (75% yield based on recovered
starting material, which could be recycled) (Scheme 8). If the
substitution reaction was performed in CH2Cl2, better substitu-
tion yields were obtained, but with a mixture of enol ether 11
and corresponding ketone. Altogether, recycling the remaining
starting product gave superior final yields of 11. The same
reaction procedure was followed with 2,4-dinitrophenylsulfuryl
chloride and allowed isolation of disulfide 12 in 17% yield (57%
yield based on recovered starting material).
Unlike our other attempts on thiophenols, thioesters, or
disulfides, McMurry coupling of benzoate 9 on adamantanone
under standard reaction conditions gave satisfactory yields of
enol ether 10 (Scheme 7).
Unsymmetrically substituted disulfide bond formation directly
from S-tert-butylsulfanyl has already been described using
(15) Terada, M.; Migata, T.; Sano, H.; Ogata, T.; Kosagi, M. Bull. Chem. Soc.
Jpn 1985, 58, 3657.
(16) Suzuki, H.; Kondo, A.; Ogawa, T. Chem. Lett. 1985, 411-412.
(17) Pastuszak, J. J.; Chimiak, A. J. Org. Chem. 1981, 46, 1868-1873.
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A R T I C L E S
Scheme 8. Final Steps of the Synthesis of 1,2-Dioxetanes 5 and
atom nucleophilic addition were then tested under chemilumi-
nescence measuring conditions, i.e., in the presence of the
chemiluminescence enhancer Sapphire in its appropriate buffer
(20 mM Tris-HCl, 1 mM MgCl2, pH 9.5).19 Increasing amounts
(0-2 equiv) of thiocholine iodide (in 10-2 M phosphate buffer,
pH 7.4) were added to a 40 µM solution of dioxetanes 6 and
Sapphire enhancer in the enhancer buffer. UV absorption at 414
nm was measured after overnight reaction at room temperature
(Figure 1a). Increase in absorption at 414 nm, reflecting an
increase in the amount of 2,4-dinitrothiophenolate, only occurred
once 1 equiv of thiocholine iodide had been added. Light
emission was then measured. In sharp contrast to the UV results,
right after addition of 0.1 equiv of thiocholine iodide to a
buffered solution of 6, flash light emission was observed (Figure
1b). The intensity of the light emission increased with the
amount of thiocholine iodide and reached a maximum inten-
sity when 0.8 equiv of thiol was added. The light could be
quantified either as the highest intensity reached (usually
3-10 s after addition of thiocholine iodide) or, more reliably,
as the total amount of light emitted in a given time lapse
(arbitrarily chosen as 800 s). Interestingly, when the same
reaction was performed with 1,2-dioxetane 5, light emission was
detected only after addition of over 1 equiv of thiocholine iodide
(Figure 1b).
Both experiments are in complete agreement with the
following mechanisms described in Scheme 9: for 1,2-dioxetane
5, thiocholine iodide first adds to the sulfur atom borne by the
electron-transferring moiety, yielding mixed disulfide 12 and
o-nitrothiophenol. Addition of a second thiocholine iodide
equivalent yields symmetric disulfide 13 and chemiluminescent
species 14. For 1,2-dioxetane 6, the additive nitro group
sufficiently activates the sulfur atom borne by the nucleo-
philic addition orienting moiety, so that chemiluminescent
species 14 is directly produced by the addition of a first
thiocholine iodide molecule. Addition of a second equivalent
of thiocholine iodide yields 2,4-dinitrothiophenol (λmax ) 402
nm) and symmetric disulfide 13. Such a surprisingly large
difference between the two 1,2-dioxetanes 5 and 6 is striking,
pinpointing the influence of the electron-withdrawing groups
borne by the nucleophilic addition-orienting moiety. Yet, an
expected ordering of the four thio-leaving groups was observed,
with 2,4-dinitrothiophenyl . 2-nitrothiophenyl . thiocoline >
thiophenyldioxetane.
6a
a (i) R ) H, 2-nitrophenyl sulfuryl chloride, THF, 2 h; R ) NO2, 2,4-
dinitrophenyl sulfuryl chloride, THF, 2 h; (ii) (PhO)3P, O3, -78 to 20 °C,
CH2Cl2.
The last step of the synthesis of 1,2-dioxetanes 5 and 6 was
the careful [2 + 2] cycloaddition of singlet oxygen 1O2 on enol
ether. In view of the great sensitivity of the disulfide bond to
highly oxidative oxygenated species, oxygen bubbling under
UV light irradiation seemed unacceptable. To monitor precisely
the amount of 1O2 delivered, we decided to use triphenyl
phosphite ozonide. This pentacoordinated phosphorane is formed
at -78 °C by the ozone-mediated oxidation of triphenyl
phosphite. Upon heating from -78 °C to room temperature, its
thermal decomposition leads to the formation of triphenyl
1
phosphate and O2.18 Using this mild and controlled singlet
oxygen source, fragile 1,2-dioxetane chemiluminescent probes
5 and 6 could be synthesized in moderate yields. No trace of
byproducts displaying oxidation at any of the two sulfur atoms
was detected. Interestingly, both 5 and 6 displayed surprisingly
high stability and could be isolated, after careful silica gel
chromatography, in 25 and 22% yield, respectively, together
with recovered starting material (61% for 11 and 67% for 12),
which could be recycled.
Chemiluminescent probes 5 and 6 were poorly soluble in
buffered aqueous media. Stock 1.2 mM solutions in either
58/42 acetone/water mixture (5) or ethyl alcohol (6) were
used. Under these conditions, both products proved per-
fectly stable for over 6 months at -20 °C. First experiments
were performed in 0.1 M phosphate buffer, pH 7.4 with
dioxetane 6. A large excess (10 equiv) of thiocholine iodide
was added to this first-generation chemiluminescent probe in
order to test the rate of the disulfide bond breaking by thiols.
The reaction was monitored by measuring the appearance of
2,4-dinitrothiophenol species via their characteristic UV absorp-
tion band at ca. 400 nm. Once thiocholine iodide was added,
within seconds the UV spectrum dramatically changed. The
absorption band at 314 nm disappeared, and a strong absorption
band was observed at 402 nm, showing addition of the
nucleophilic thiol to the disulfide and release of the 2,4-
dinitrothiophenolate ion.
The luminescence of 6 was then measured in the presence of
AChE and acetylthiocholine iodide. Sapphire and its buffer both
inhibited AChE, so only endpoint measurements were possible.
To estimate the detection limits of this chemiluminescent probe,
acetylthiocholine iodide was incubated for 30 min with various
amounts of E. electricus AChE [in its tetrameric form, in EIA
buffer, namely 0.1 M phosphate buffer (pH 7.4) containing 0.15
M NaCl, 10-3 M EDTA, 0.1% bovine serum albumin, and
(18) (a) Murray, R. W.; Kaplan, M. L. J. Am. Chem. Soc. 1969, 91, 5358-
5364. (b) Stephenson, L. M.; Zielinski, M. B. J. Am. Chem. Soc. 1982,
104, 5819-5820.
(19) Aqueous environments rapidly reduce the chemiluminescent signal intensity
of 1,2-dioxetane substrates by water-induced quenching. Chemilumines-
cence enhancers (quaternary ammonium with fluorescent relay either as
micelles or polymers) increase the emission efficiency by partitioning the
water from the site of chemiluminescent signal production. Cf. (a) Kohne,
D. E. US patent WO9705209, 1997; Chem. Abstr. 1997, 126, 222603. (b)
Akhaven-Tafti, H.; Arghavani, Z.; Eickholt, R.; Rashid, K. US patent
WO9615122, 1996; Chem. Abstr. 1996, 125, 479234. (c) Schaap, A. P.;
Akhavan-Tafti, H. US patent WO9007511, 1990; Chem. Abstr. 1991, 114,
181726.
Regioselectivity of this disulfide bond cleavage and the
capacity of dioxetanes 6 to emit light when subject to a sulfur
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Sabelle et al.
Figure 1. (a) Effect of increasing amounts of thiocholine iodide on the absorbance at 414 nm when incubated overnight with dioxetane 6 (b). The addition
of thiocholine iodide (10 µM to 80 µM) on 6 (40 µM) was carried out at 25 °C pH 9.0, in 3.3 × 10-3 M phosphate, 13.3 × 10-3 M Tris-HCl, and in the
presence of 6.6 × 10-4 M MgCl2 and 1/3 v/v enhancer Sapphire. (b) Effect of increasing amounts of thiocholine iodide on the light emission in the presence
of dioxetane 5 (9) or 6 (b). The reaction of thiocholine iodide (10-80 µM) with 5 or 6 (40 µM) was carried out at 25 °C pH 9.0, in 3.3 × 10-3
phosphate, 13.3 × 10-3 M Tris-HCl, and in the presence of 6.6 × 10-4 M MgCl2 and 1/3 v/v enhancer Sapphire.
M
Scheme 9. Proposed Mechanisms for the Addition of Thiocholine Iodide on Chemiluminescent Probes 5 and 6
0.01% sodium azide], and the amount of light emitted was
measured. As depicted in Figure 2, the amount of light emitted
can be directly related to the AChE concentration.
The background light emission is due to the uncatalyzed
hydrolysis of acetylthiocholine iodide, so a compromise has to
be found between (i) an optimal concentration of acetylthio-
choline iodide, to be as close as possible to the enzymatic Vmax
,
and (ii) the uncatalyzed hydrolysis rate of acetylthiocholine
iodide, directly related to its initial concentration. Under
optimized enzymatic reaction conditions, the detection limit
(defined as the background reaction + 3SD) could be estimated
to be 0.1 fM, which, under the reaction conditions used,
corresponds to 2.5 × 10-18 mol of enzyme (ca. the same
detection limit as for the colorimetric reaction).
The first way to increase the sensitivity of the assay would
be to increase the enzymatic reaction time, once we have
checked, as depicted in Figure 3, that the amount of light emitted
is proportional to the enzymatic reaction time.
Since immunoassays need short staining times, we turned to
another parameter which could lower the detection limit: the
use of a stronger luminescence enhancer. Interestingly, when
Sapphire II enhancer [poly(vinylbenzyltributylammonium chlo-
ride)] was used, a new phenomenon was observed: flash
luminescence was replaced by glow luminescence, with light
production lasting for minutes. Once again, the sensitivity of
the assay was directly related to the background acetylthiocho-
line iodide hydrolysis. To minimize this limitation, the amount
of light emitted in a two-minute period was measured once glow
Figure 2. Emission of light after 30-min incubation of acetylthiocholine
iodide (0.75 mM) and various amounts of E. electricus AChE (0.25-83.3
fM). The enzymatic reaction was performed at 25 °C in 2.5 × 10-2
M
phosphate buffer pH 7.4, containing 0.04 M NaCl, 2.5 × 10-4 M EDTA,
0.025% bovine serum albumin, and 0.0025% sodium azide. Light measure-
ment was performed with 40 µM 1,2-dioxetane 6, at 25 °C pH 9.0, in 3.3
× 10-3 M phosphate, 13.3 × 10-3 M Tris-HCl, and in the presence of 6.6
× 10-4 M MgCl2 and 1/3 v/v enhancer Sapphire. Amount of light emitted
was recorded at 800 s. The dotted line corresponds to the background signal
observed without enzyme.
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Chemiluminescent Probes for Cholinesterase Activity
A R T I C L E S
silica gel 60 (0.02-0.04 mm). TLC was performed with fluorescent
Merck F254 glass plates. NMR spectra were recorded on a Brucker
AC-300 (300.15 MHz for 1H NMR and 75.4 MHz for 13C NMR).
Chemical shifts (δ) are given in ppm and the coupling constant J is
expressed in hertz. MS were obtained with a Finnigan-Mat 4600
quadrupole system.
3-Iodobenzoic Acid Ethyl Ester (7). Ethyl 3-bromobenzoate (1.5
mL, 9.37 mmol) was added to a suspension of 23.3 g (140 mmol) of
KI and 8.92 g (47 mmol) of CuI in 56 mL of dry HMPA. The reaction
mixture was heated to 155 °C for 3 days. After cooling to room
temperature, the reaction mixture was added to 200 mL of brine. The
aqueous phase was washed thrice with diethyl ether. The combined
organic phases were dried over MgSO4 and concentrated under reduced
pressure. The crude reaction mixture was chromatographed on silica
gel (hexane/ethyl acetate 95/5), yielding 2.38 g (92%) iodobenzoate 7
as a colorless oil.
Figure 3. Influence of the time of incubation of acetylthiocholine iodide
(0.75 mM) and AChE (0.25 fM) on the luminescence signal. Enzymatic
reaction was performed at 25 °C, in 2.5 × 10-2 M phosphate buffer pH
7.4, containing 0.04 M NaCl, 2.5 × 10-4 M EDTA, 0.025% bovine serum
albumin, and 0.0025% sodium azide for increasing times (23-115 min)
before light recording. Light was measured with 40 µM 1,2-dioxetane 6, at
25 °C pH 9.0, in 3.3 × 10-3 M phosphate, 13.3 × 10-3 M Tris-HCl, and
in the presence of 6.6 × 10-4 M MgCl2 and 1/3 v/v enhancer Sapphire.
The light emitted was recorded at 720 s (12 min).
1H NMR (300 MHz, CDCl3): δ 1.27 (t, J ) 9.5 Hz, 3H), 4.24 (q,
J ) 9.5 Hz, 2H), 7.01 (t, J ) 8.0 Hz, 1H), 7.70 (dt, J ) 8.0 and 2.5
Hz, 1H), 7.85 (dt, J ) 8.0 and 1.5 Hz, 1H), 8.23 (t, J ) 1.5 Hz, 1H).
13C NMR (75.4 MHz, CDCl3): δ 14.35, 61.26, 93.87, 128.62, 130.05,
132.28, 138.25, 141.51, 164.72. MS (CI, NH3): m/z 294 (MH+), 311
(MNH4+).
(tert-Butylsulfanyl)tributyltin (8). Under argon atmosphere, 4.1 mL
(15.1 mmol) of tributyltin chloride was added to a mixture of 1.70 mL
(15.1 mmol) of tert-butylthiol and 2.53 mL (18.1 mmol) of triethylamine
in 100 mL of dry CCl4. After overnight agitation at room temperature,
the reaction mixture was filtered on a Celite pad and washed
successively with acetic acid (100 mL of a 5% v/v aqueous solution)
and water (100 mL). The organic phase was dried over MgSO4, and
the solvents were evaporated under vacuum. The crude reaction mixture
(5.7 g, 98% yield of 18 as a white solid) proved sufficiently pure to be
used as such for the next step.
Figure 4. Light emission after 45-min incubation of acetylthiocholine iodide
(1.25 mM) and various amounts of E. electricus AChE (0.025-250 fM).
The enzymatic reaction was performed at 25 °C, in 2.5 × 10-2 M phosphate
buffer pH 7.4, containing 0.04 M NaCl, 2.5 × 10-4 M EDTA, 0.025%
bovine serum albumin, and 0.0025% sodium azide. Light was measured
with 80 µM 1,2-dioxetane 6, at 25 °C pH 9.0, in 3.3 × 10-3 M phosphate,
13.3 × 10-3 M Tris-HCl, and in the presence of 6.6 × 10-4 M MgCl2 and
1/3 v/v enhancer Sapphire II. The light emitted during 2 min was recorded
after a 2-min delay. Dotted line corresponds to the background signal
observed without enzyme.
1H NMR (300 MHz, CDCl3): δ 0.76 (bt, 3JH-H ) 7.0 Hz, 9H), 0.97-
3
3
1.02 (bdd, JH-H ) 7.0 and 8.0 Hz, 6H), 1.12-1.23 (bq, JH-H ) 7.0
Hz, 6H), 1.28 (bs, 9H), 1.37-1.49 (m, 6H). 13C NMR (75.4 MHz,
CDCl3): δ 13.51 (3C), 14.31 (1s + 2d, 1JSn-C ) 310 and 330 Hz, 3C),
luminescence started. Under optimized conditions (Figure 4),
the detection limit was estimated as 0.01 fM, which corresponds,
under the reaction conditions, to 2.5 × 10-19 mol.
2
26.97 (1s + 2d, JSn-C ) 60 and 63 Hz, 3C), 28.64 (3C), 36.60 (3C),
43.11.
3-(tert-Butylsulfanyl)benzoic Acid Ethyl Ester (9). In a glovebox,
purged under N2, 83.7 mg (0.072 mmol) of Pd(PPh3)4 was added to a
solution of ethyl 3-iodobenzoate 7 (200 mg, 0.72 mmol) in 20 mL of
dry toluene. To this bright yellow solution was added a solution of
302 mg of (tert-butylsulfanyl)tributyl tin 8 dissolved in 5 mL of toluene.
The reaction mixture was then refluxed for 48 h under an argon
atmosphere. After cooling to room temperature, 50 mL of diethyl ether
and 50 mL of a 10% aqueous KF solution were added. The aqueous
phase was extracted thrice with diethyl ether (100 mL). The combined
organic phases were dried over MgSO4 and concentrated under reduced
pressure. The crude reaction mixture was then chromatographed on
silica gel (hexane/ethyl acetate 95/5), yielding 128.5 mg (75%) of tert-
butylsulfanyl 9 as a pale yellow oil.
1H NMR (300 MHz, CDCl3): δ 1.30 (s, 9H), 1.40 (t, J ) 9.5 Hz,
3H), 4.39 (q, J ) 9.5 Hz, 2H), 7.33 (t, J ) 7.5 Hz, 1H), 7.64 (bd, J )
7.5 Hz, 1H), 8.04 (bd, J ) 7.5 Hz, 1H), 8.21 (bs, 1H). 13C NMR (75.4
MHz, CDCl3): δ 14.41, 31.04(3C), 46.11, 61.23, 128.51, 129.86,
131.02, 133.42, 138.25, 141.68, 166.19. MS (CI, NH3): m/z 256
(MNH4+).
2-[1-(3-(tert-Butylsulfanyl)phenyl)-1-ethoxymethylene]tricyclo-
[3.3.1.13,7]decane (10). In a glovebox, under N2 atmosphere, 14.1 mL
of LiAlH4 (1 M solution in ether, 14.1 mmol) was added to a cooled
(0 °C) suspension of 4.36 g of TiCl3 (28.3 mmol) in 35 mL of dried
THF. After 10 min at 0 °C, 2.37 mL of triethylamine (16.9 mmol) was
added, and the suspension was refluxed for 1 h. A mixture of 532 mg
of adamantanone (3.53 mmol) and 675 µL of ethyl benzoate 9 (2.83
mmol) in 8 mL of THF was then added dropwise for 2.5 h. The reaction
mixture was refluxed overnight, cooled to room temperature, and
Conclusion
In conclusion, we have synthesized a new chemiluminescent
1,2-dioxetane probe 6 for the detection of AChE activity. Testing
this first-generation probe for use in AChE-based immunoassays,
in microtiter plates, we have been able to reach a 0.01 fM
detection limit, ca. 10 times lower than with the colorimetric
assays. We have shown, for the first time, that not only
m-oxybenzoate anions but also m-mercaptobenzoate anions can
be used in intramolecular CIEEL systems. The main drawback
of our probe is the required use of a chemiluminescent enhancer
that fully inhibits AChE, preventing any continuous light
measurements. These encouraging first results have prompted
us to synthesize new chemiluminescent probes containing either
tethered fluorescers in their very structure, to have an intramo-
lecular fluorescence/luminescence relay,20 or an O-S bond
instead of the disulfide bond, to enhance the luminescence
quantum yield.
Experimental Section
Synthesis. General Remarks. Reagents were from Aldrich. All
solvents were distilled before use, and reactions were performed under
N2 atmosphere. All chromatography (flash) was performed with Merck
(20) (a) Schapp, A. P.; Akhavan-Tafti, H. US Patent, US 5616729, 1997; Chem.
Abstr. 1997, 126, 317373. (b) de Silva, R. K.PhD Dissertation, Wayne State
University, Detroit, MI, 1989.
9
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A R T I C L E S
Sabelle et al.
partitioned between diethyl ether (200 mL) and water (200 mL). The
aqueous phase was extracted thrice with diethyl ether (200 mL), and
the combined organic phases were dried over MgSO4 and concentrated
under reduced pressure. The crude reaction mixture was chromato-
graphed on silica gel (hexane/ethyl acetate 98/2), yielding 604 mg (60%)
of McMurry condensation enol ether 10 as a colorless oil.
1H NMR (300 MHz, CDCl3): δ 1.15 (t, J ) 9.0 Hz, 3H), 1.31 (s,
9H), 1.60-1.99 (m, 12H), 2.63 (bs, 1H), 3.29 (bs (1H), 3.47 (q, J )
9.0 Hz, 2H), 7.31-7.37 (m, 2H), 7.44 (bd, J ) 7.5 Hz, 1H), 7.51 (bs,
1H). 13C NMR (75.4 MHz, CDCl3): δ 15.19, 28.48 (2C), 30.46, 31.06
(3C), 32.53, 37.32, 39.06 (2C), 39.32 (2C), 46.02, 64.74, 128.02, 128.20,
129.40, 129.67, 132.31, 135.14, 136.39, 138.53. MS (CI, NH3): m/z
357 (MH+).
2-{Ethoxy[3-(2-nitrophenyldisulfanyl)phenyl]methylene}tricyclo-
[3.3.1.13,7]decane (11). 2-Nitrobenzenesulfenyl chloride (65 mg, 0.34
mmol) was added to a solution of 116 mg (0.325 mmol) of enol ether
10 in 3 mL of dry THF. After 2 h at room temperature, the solvent
was removed under vacuum. The crude reaction mixture was chro-
matographed on silica gel (hexane/ethyl acetate/Et3N 95/5/0.1), yielding
75 mg (65%) recovered starting material 10 and then 39 mg (26%)
disulfide 11 as a colorless oil.
1H NMR (300 MHz, CDCl3): δ 1.11 (t, J ) 9.0 Hz, 3H), 1.66-
1.94 (m, 12H), 2.51 (bs, 1H), 3.23 (bs, 1H), 3.42 (q, J ) 9.0 Hz, 2H),
7.21-7.41 (m, 5H), 7.63 (td, J ) 1.0 and 7.5 Hz, 1H), 8.15 (dd, J )
1.0 and 8.5 Hz 1H), 8.31 (dd, J ) 1.0 and 8.5 Hz, 1H). 13C NMR
(75.4 MHz, CDCl3): δ 15.19, 28.30 (2C), 30.55, 32.44, 37.19, 39.0
(2C), 39.23 (2C), 65.0, 126.21, 126.50, 127.50, 127.93, 128.44, 129.06,
133.58, 134.24, 137.20, 137.40. MS (CI, NH3): m/z 454 (MH+), 471
(MNH4+).
2-{Ethoxy[3-(2,4-dinitrophenyldisulfanyl)phenyl]methylene}-
tricyclo[3.3.1.13,7]decane (12). 2,4-Dinitrobenzenesulfenyl chloride
(625 mg, 2.66 mmol) was added to a solution of 949 mg (2.66 mmol)
of enol ether 10 in 7 mL of dry THF. After 2 h at room temperature,
the solvent was removed under vacuum. The crude reaction mixture
was chromatographed on silica gel (hexane/ethyl acetate/Et3N 95/5/
0.1), yielding 666 mg (70%) of recovered starting material 10 and then
225 mg (17%) disulfide 12 as a pale yellow oil.
1H NMR (300 MHz, CDCl3): δ 1.12 (t, J ) 9.0 Hz, 3H), 1.70-
1.96 (m, 12H), 2.53 (bs, 1H), 3.24 (bs, 1H), 3.42 (q, J ) 9.0 Hz, 2H),
7.24-7.39 (m, 3H), 7.43 (bs, 1H), 8.44 (bs, 2H), 9.13 (t, J ) 1.0 Hz,
1H). 13C NMR (75.4 MHz, CDCl3): δ 15.20, 28.28 (2C), 30.59, 32.50,
37.15, 38.99 (2C), 39.32 (2C), 65.11, 121.65, 127.15, 127.57, 128.69,
128.97, 129.36 (2C), 133.47, 134.05, 137.79, 140.84, 145.90. MS (CI,
NH3): m/z 499 (MH+), 516 (MNH4+).
4-Ethoxy-4-[[3-(2-nitrophenyldisulfanyl)phenyl]spiro(1,2-di-
oxetane-3,2′-tricyclo[3.3.1.13,7]decane) (5). Ozone was gently bubbled
in a cooled (-78 °C) solution of triphenyl phosphite (32.7 µL, 0.12
mmol) in 8 mL of dry CH2Cl2 until the appearance of a light blue
coloration. The reaction medium was then purged with argon until the
blue coloration disappeared. Enol ether 11 (37.7 mg; 0.083 mmol),
dissolved in 2 mL of dry CH2Cl2 was added at -78 °C. The reaction
mixture was gently brought to room temperature. Solvents were
removed under reduced pressure, and the crude reaction product was
chromatographed on silica gel (hexane/ethyl acetate 90/10), yielding
first 23 mg (61%) of recovered starting material 11 and then 10 mg
(25%) of dioxetane 5 as a pale yellow oil.
128.47, 128.91, 131.28, 133.81, 135.29, 136.42, 136.81, 145.20. UV
[λ, nm (ꢀ), in EtOH]: 347 (2686). MS (CI, NH3): m/z 503 (MNH4+).
4-Ethoxy-4-[[3-(2,4-dinitrophenyldisulfanyl)phenyl]spiro(1,2-di-
oxetane-3,2′-tricyclo[3.3.1.13,7]decane) (6). The same reaction pro-
cedure as for the synthesis of dioxetane 5 was performed with 49.3 µL
of triphenyl phosphite (0.188 mmol) and 62.5 mg of enol ether 21 (0.126
mmol). Flash chromatography on silica gel (hexane/ethyl acetate 90/
10) yielded first 42 mg (67%) of recovered starting material 12 and
then 14.5 mg (22%) dioxetane 6 as a yellow oil.
1H NMR (300 MHz, CDCl3): δ 0.82-0.91 (m, 2H), 1.11-1.15 (m,
1H), 1.23 (t, J ) 9.5 Hz, 3H), 1.38-1.43 (m, 1H), 1.50-1.82 (m, 8H),
1.92 (bs, 1H), 3.05 (bs, 1H), 3.15 (bquint, J ) 9.5 Hz, 1H), 3.53 (quint,
J ) 9.5 Hz, 1H), 7.41 (t, J ) 7.5 Hz, 1H), 7.52 (d, J ) 7.5 Hz, 1H),
7.60 (m, 1H), 7.80 (m, 1H), 8.43 (bs, 2H), 9.16 (bs, 1H). 13C NMR
(75.4 MHz, CDCl3): δ 15.30, 25.99, 26.06, 29.78, 31.68, 31.91, 32.20,
33.08, 33.33, 34.86, 36.38, 58.51, 95.40, 111.03, 121.89, 127.54, 128.87,
129.32, 129.60, 134.31, 137.66, 145.27, 145.96. UV [λ, nm (ꢀ), in
EtOH]: 304 (9010). MS (CI, NH3): m/z 548 (MNH4+).
Luminescence Assays. General. Enhancers and their buffers were
from TROPIX (enhancer buffer: 20 mM Tris-HCl, 1 mM MgCl2, pH
9.5). The G4 form of E. electricus AChE was purified and stored as
described elsewhere.21 EIA buffer comprised 0.1 M phosphate buffer
(pH 7.4) containing 0.15 M NaCl, 10-3 M EDTA, 0.1% bovine serum
albumin, and 0.01% sodium azide. Luminescence emission was
recorded on a DYNATECH ML 3000 luminometer using Microlite II
microtiter plates. UV spectra were recorded on an HP 8452 diode array
spectrophotometer. Colorimetric assays were recorded on a LAB-
SYSTEM multiscan bichromatic plate reader at 414 nm.
Direct Colorimetric and Luminescence Assay. In a microtiter plate
were introduced successively 50 µL of a thiocholine iodide solution in
10-2 M phosphate buffer pH 7.4 (4.8 × 10-4 to 6 × 10-5 M), 50 mL
of EIA buffer, 100 µL of Sapphire enhancer, and 100 µL of
1.2-dioxetane 5 or 6 (1.2 × 10-4 M in a 1/9 mixture of EtOH and
enhancer buffer). For the colorimetric assay, the absorbance at 414 nM
of each well was measured after overnight, room-temperature incuba-
tion. For the luminescence assay, light emission was measured directly
after addition of 1,2-dioxetane 5 or 6.
Enzymatic Assays. In a microtiter plate were introduced successively
75 µL of a thiocholine iodide solution in 10-2 M phosphate buffer pH
7.4 (1.5 × 10-3 M for the assays with enhancer Sapphire, 2 × 10-3
M
for the assays with enhancer Sapphire II) and 25 mL of E. electricus
AChE as its G4 isoform, purified and stored as described elsewhere21
(dilution range from 10 to 0.001 Ellman units) in EIA buffer. After a
30-min enzymatic reaction, 100 µL of enhancer Sapphire or Sapphire
II and 100 µL of 1,2-dioxetane 6 (1.2 × 10-4 M in a 1/9 mixture of
EtOH and enhancer buffer for use with enhancer Sapphire, 2.4 × 10-4
M in a 2/8 mixture of EtOH and enhancer buffer for use with enhancer
Sapphire II) were added. Light emission was measured directly after
addition of 1,2-dioxetane 6.
Acknowledgment. S.S. was financially supported by SPI Bio.
P.-Y.R. was financially supported by De´le´gation Ge´ne´rale pour
l’Armement. The authors are indebted to Dr. J. C. Nicolas
(Universite´ de Montpellier) for the early suggestion to use the
disulfide chemiluminescent probe for AChE detection.
1H NMR (300 MHz, CDCl3): δ 0.80-0.89 (m, 2H), 1.08-1.13 (m,
1H), 1.20 (t, J ) 9.5 Hz, 3H), 1.38-1.43 (m, 1H), 1.55-1.84 (m, 8H),
1.95 (bs, 1H), 3.03 (bs, 1H), 3.15 (bquint, J ) 9.5 Hz, 1H), 3.52 (quint,
J ) 9.5 Hz, 1H), 7.38 (bt, J ) 8.5 Hz, 2H), 7.53 (bd, J ) 9.0 Hz, 1H),
7.62 (bt, J ) 7.5 Hz, 2H), 7.65-7.80 (m, 1H), 8.16 (bd, J ) 7.5 Hz,-
1H), 8.31 (bd, J ) 8.5 Hz,1H). 13C NMR (75.4 MHz, CDCl3): δ 15.42,
25.62, 25.77, 29.05, 29.44, 31.35, 31.53, 31.93, 32.65, 32.91, 34.48,
36.11, 39.01, 58.07, 95.05, 110.86, 125.88, 126.33, 127.06, 127.64,
Supporting Information Available: Spectra for compounds
5 and 6. This material is available free of charge via the Internet
at http://pubs.acs.org.
JA0171299
(21) (a) Massoulie´, J.; Bon, S. Eur. J. Biochem. 1976, 68, 531-539. (b) Pradelles,
P.; Grassi, J.; Maclouf, J. Anal. Chem. 1985, 57, 1170-1173
9
4880 J. AM. CHEM. SOC. VOL. 124, NO. 17, 2002