Effect of surfactants on the chemiluminescence of acridinium dimethylphenyl ester labels and their conjugates

Article abstract of DOI:10.1039/c1ob05543g

Chemiluminescent acridinium dimethylphenyl esters, containing two methyl groups flanking the phenolic ester bond, display excellent chemiluminescence stability and are used as labels in automated immunoassays for clinical diagnostics. Light emission from these labels is triggered with alkaline peroxide in the presence of the cationic surfactant cetyltrimethylammonium chloride. Under these conditions, light emission is rapid and is complete in <5 s. In the present study we examined the effect of various surfactants on light emission from acridinium dimethylphenyl ester labels and their conjugates containing hydrophilic linkers derived either from hexa(ethylene)glycol or a sulfobetaine zwitterion. Sulfobetaine zwitterions are very polar and incorporation of these functional groups in acridinium dimethyphenyl esters and their conjugates represents a new approach to improving the aqueous solubility of these chemiluminescent labels. Our results indicate that in general, surfactants affect light emission from these labels and their conjugates by two discrete mechanisms. Cationic surfactants, but not anionic or non-ionic surfactants, accelerate overall light emission kinetics and a more modest effect is observed with zwitterionic surfactants. Surfactants also enhance total light output and the magnitude of this enhancement is maximal for cationic surfactants and a sulfobetaine zwitterionic surfactant. These observations are the first to clearly delineate the role of the surfactant on the chemiluminescence reaction pathway of acridinium esters and can be rationalized based on known effects of surfactant aggregates on bimolecular and unimolecular reactions. The Royal Society of Chemistry 2011.

Full text of DOI:10.1039/c1ob05543g

View Article Online / Journal Homepage / Table of Contents for this issue
Organic &
Biomolecular
Chemistry
Dynamic Article Links
Cite this: Org. Biomol. Chem., 2011, 9, 5092
www.rsc.org/obc
PAPER
Effect of surfactants on the chemiluminescence of acridinium dimethylphenyl
ester labels and their conjugates†
Anand Natrajan,* David Sharpe and David Wen
Received 7th February 2011, Accepted 12th April 2011
DOI: 10.1039/c1ob05543g
Chemiluminescent acridinium dimethylphenyl esters, containing two methyl groups flanking the
phenolic ester bond, display excellent chemiluminescence stability and are used as labels in automated
immunoassays for clinical diagnostics. Light emission from these labels is triggered with alkaline
peroxide in the presence of the cationic surfactant cetyltrimethylammonium chloride. Under these
conditions, light emission is rapid and is complete in <5 s. In the present study we examined the effect
of various surfactants on light emission from acridinium dimethylphenyl ester labels and their
conjugates containing hydrophilic linkers derived either from hexa(ethylene)glycol or a sulfobetaine
zwitterion. Sulfobetaine zwitterions are very polar and incorporation of these functional groups in
acridinium dimethyphenyl esters and their conjugates represents a new approach to improving the
aqueous solubility of these chemiluminescent labels. Our results indicate that in general, surfactants
affect light emission from these labels and their conjugates by two discrete mechanisms. Cationic
surfactants, but not anionic or non-ionic surfactants, accelerate overall light emission kinetics and a
more modest effect is observed with zwitterionic surfactants. Surfactants also enhance total light
output and the magnitude of this enhancement is maximal for cationic surfactants and a sulfobetaine
zwitterionic surfactant. These observations are the first to clearly delineate the role of the surfactant on
the chemiluminescence reaction pathway of acridinium esters and can be rationalized based on known
effects of surfactant aggregates on bimolecular and unimolecular reactions.
increase their aqueous solubility, lower their non-specific binding
Introduction
and to increase their quantum yields.2 Light emission from these
Chemiluminescence has emerged to be a dominant detection tech-
labels is triggered in two steps. An initial treatment with acid
rapidly converts the non-chemiluminescent pseudobase6 (water
adduct) 1 (Fig. 2) to the chemiluminescent acridinium ester 2
(Fig. 2). Subsequent reaction of the acridinium ester with alkaline
hydrogen peroxide generates chemiluminescence. The cationic sur-
factant cetyltrimethylammonium chloride which enhances overall
light output7 is included in the triggering reagents. Under these
conditions, light emission from acridinium dimethylphenyl esters
is quite rapid and is essentially complete in <5 s.2
Excited state acridone 6 (Fig. 2) is believed to be the light
emitting species in the chemiluminescence reaction of acridinium
phenyl esters and is presumably formed by addition of hydroper-
oxide ions to C-9 of the acridinium ring followed by scission of the
phenolic ester bond.8,9 A high energy dioxetanone intermediate 5
(Fig. 2) has been proposed in the reaction pathway to the excited
state acridone,8,9 but computational studies have suggested that its
direct formation from the dioxetane 4 (Fig. 2) is energetically more
favorable.10 This latter study10a also identified the formation of this
dioxetane as the slow step in the overall reaction sequence but this
hypothesis has not been experimentally verified. Detailed kinetics
parameters for the chemiluminescence of various acridinium
esters as well as fluorescence measurements of the corresponding
nology in the clinical diagnostics industry and chemiluminescent
labels are widely used in automated instruments.1 Among the
different chemiluminescent labels, acridinium phenyl esters are
especially attractive because of their high sensitivity with detection
limits in the attomole range. Moreover, they exhibit fast light
emission using simple triggering reagents. The relatively small
size of acridinium esters is also beneficial for minimizing steric
interference in binding reactions.2–5 Chemiluminescent acridinium
ester labels containing two methyl groups flanking the phenolic
ester bond2–5 (Fig. 1) are used in automated, immunochemistry
instruments for clinical diagnostics. These acridinium ester labels
demonstrate significantly improved chemiluminescence stability
compared to unsubstituted, acridinium phenyl esters at physiolog-
ical pH.3 In addition, acridinium dimethylphenyl esters are fairly
easy to synthesize with a variety of structural modifications to
Siemens Healthcare Diagnostics, Advanced Technology and Pre-
Development, 333 Coney Street, East Walpole, MA 02032, USA. E-mail:
anand.natrajan@siemens.com; Fax: +1-508-660-4591; Tel: +1-508-660-
4582
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c1ob05543g
5092 | Org. Biomol. Chem., 2011, 9, 5092–5103
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
Fig. 1 Structures of acridinium ester labels 1a and 1b, theophylline conjugates 2a and 2b and, NHS ester labels 3a and 3b used for protein labeling.
-
Fig. 2 Simplified reaction pathway for chemiluminescence from acridinium esters. R = –CH2CH2CH2SO3 for compounds 1a, 1b, 2a, 2b and protein
conjugates of 3a and 3b. Formation of both dioxetane 4 and dioxetanone 5 involve dispersal of negative charge in the transition states of these reactions.
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 5092–5103 | 5093
©

View Article Online
acridones in various solvents have also been recently reported10b,c
and they support the notion that excited state acridone is the
primary emitter. Decomposition of the dioxetane 4 or dioxetanone
5 is postulated to occur by electron transfer from the acridine
nitrogen to the peroxide bond9–11 by a mechanism analogous to
the CIEEL (chemically initiated electron-exchange luminescence)
mechanism proposed by Schuster.12 A simplified version of the
reaction pathway leading to chemiluminescence from acridinium
esters is illustrated in Fig. 2.
that present in compound 1b thus represents a complementary
approach towards increasing the aqueous solubility and lowering
the non-specific binding of acridinium ester labels.
The syntheses of compounds 1a–3a have been described
previously.2,5 Syntheses of compounds 1b, 2b and 3b containing the
zwitterionic sulfobetaine linker are illustrated in Fig. 3 and were
accomplished in a straightforward manner using commercially
available reagents. The zwitterionic linker iv itself was synthe-
sized in three steps from N,N-bis(3-aminopropyl)methylamine
as described in the Experimental section. The primary amines
in N,N-bis(3-aminopropyl)methylamine i were first converted to
the benzyl carbamates, following which the protected derivative
ii was N-alkylated at the tertiary amine with 1,3-propane sultone
to give compound iii. Deprotection of the primary amines in iii
gave the zwitterionic linker iv. The linker iv was next coupled
to acridinium ester v4,5 to give compound 1b which was purified
by preparative HPLC. Conversion of 1b to the theophylline
conjugate 2b was accomplished in one step by coupling to commer-
cially available 8-carboxypropyltheophylline using (benzotriazol-
1-yl-oxy)tris(dimethylamino)phosphonium hexafluorophosphate
(BOP) followed by HPLC purification. Conversion of 1b to
the NHS ester label 3b was accomplished by first condensing
1b with glutaric anhydride followed by activation of the re-
sulting carboxylate derivative with N,N,N¢,N¢-tetramethyl-O-(N-
succinimidyl)uronium tetrafluoroborate (TSTU). Compound 3b
was purified by HPLC prior to protein labeling.
Protein conjugates of the labels 3a and 3b were prepared,
as described in the experimental section, using three different
proteins; a murine anti-TSH monoclonal antibody with an acidic
pI = 5.6 (TSH = Thyroid Stimulating Hormone); a murine anti-
HBsAg monoclonal antibody with a pI = 7 (HBsAg = Hepatitis B
Surface Antigen) and egg white avidin with a basic pI = 10.5.18 All
three labels displayed similar reactivity towards the three proteins
and the extent of label incorporation was very similar as described
in the Experimental section. Using an input of 10 equivalents
of the acridinium ester labels 3a and 3b, approximately 5 labels
were incorporated in each protein as measured by MALDI-TOF
(Matrix-Assisted Laser Desorption Ionization-Time of Flight)
mass spectrometry.
In
a previously reported study, the cationic surfactant
cetyltrimethylammonium chloride was observed to enhance over-
all light output from an acridinium dimethylphenyl ester con-
taining an N-methyl group.7 Similar effects of surfactants in
enhancing the luminescence of other chemiluminescent labels such
as luminol,13 peroxyoxalates14 and dioxetanes15 have also been
observed. In a study that examined the effect of surfactants on
the chemiluminescence of acridinium phenyl ester labels lacking
the dimethyl groups, antibody conjugates of these labels were
observed to exhibit the greatest increase in chemiluminescence
in the presence of the non-ionic surfactant triton X-100, whereas
cetyltrimethylammonium chloride was most effective for albumin
conjugates.16
In the current study, we examined the effect of cationic,
zwitterionic, anionic and non-ionic surfactants on light emission
from two hydrophilic acridinium dimethylphenyl ester labels as
well as their conjugates (Fig. 1) that are currently used in
automated immunoassays. The purpose of this study was mainly
to elucidate the role of the surfactant on the chemiluminescence
from these hydrophilic labels especially pertaining to the reaction
pathway illustrated in Fig. 2. We felt that a better understanding of
the role of the surfactant was needed to determine whether there is
scope for further enhancement of the chemiluminescence of these
labels. From a practical point of view, increased light output from
these labels would be beneficial in improving assay sensitivity of
clinically important analytes.
Results and discussion
Acridinium esters and conjugates
Hydrophilic acridinium dimethylphenyl esters (Fig. 1) dis-
play excellent chemiluminescence stability and low non-specific
binding,2,5 and are used as labels in automated immunoassays. In
the current study, the free labels 1a and 1b in Fig. 1 were used
for the preparation of small molecule theophylline conjugates 2a
and 2b. Protein conjugates were prepared using the NHS (N-
hydroxysuccinimidyl) ester derivatives 3a and 3b. Compound 1a
with an unsubstituted N-sulfopropyl acridinium ring contains
a hydrophilic linker derived from hexa(ethylene)glycol which is
attached para to the phenolic ester bond. This linker increases
aqueous solubility of the label and facilitates conjugate synthesis
and protein labeling.5 Compound 1b is structurally distinct from
1a and contains a hydrophilic linker with a sulfobetaine zwitterion.
Zwitterions are highly hydrophilic and appear to complement
poly(ethylene)glycol in their ability to reduce non-specific binding
of proteins.17 Recent studies have shown that surfaces functional-
ized with zwitterions such as sulfobetaines and carboxybetaines
are extremely resistant to protein adsorption, fouling and biofilm
formation.17 The incorporation of a zwitterionic linker such as
Surfactants and light measurement protocol
Previous studies7,16 that examined the effect of surfactants on
acridinium ester chemiluminescence noted an enhancement of
chemiluminescence in the presence of surfactants but the mecha-
nism leading to this enhancement was not defined clearly. Micellar
catalysis of bimolecular reactions largely results from increased
local concentrations of the reactants in a small volume of the
micellar phase.19 An important property of ionic micelles, that
makes them effective catalysts of bimolecular reactions, is their
ability to bind various substrates and attract oppositely charged
reactive ions to their surfaces. The increased local concentration
of the two reactants is manifested by an increase in the observed
reaction rate. Surfactant aggregates also have significant effects on
the rates of unimolecular reactions such as the decarboxylation
of 6-nitrobenzisoxazole-3-carboxylate, the intramolecular cycliza-
tion reactions of ortho-haloalkyl-substituted phenoxides and
1,2-elimination reactions.20,21 All these reactions involve charge
dispersal in their transition states leading to products and are
5094 | Org. Biomol. Chem., 2011, 9, 5092–5103
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
Fig. 3 Synthetic scheme for 1b, 2b and 3b. Reagents: (a) N-(benzyloxycarbonyloxy)succinimide, chloroform; (b) 1,3-propane sultone, ethyl acetate; (c)
33% hydrogen bromide in acetic acid; (d) 0.25 M sodium bicarbonate/dimethyl formamide; (e) glutaric anhydride, diisopropylethylamine, water/methanol;
(f) TSTU, diisopropylethylamine, water/dimethyl formamide; (g) 8-carboxypropyltheophylline, BOP reagent, diisopropylethylamine, dimethyl sulfoxide.
catalyzed by various cationic and zwitterionic surfactants resulting
from reduced medium polarity of the micellar environment.20,21
Light emission from acridinium dimethylphenyl esters that are
used as chemiluminescent labels in automated immunoassays is
triggered by the sequential addition of equal volumes (0.3 mL)
of two reagents. An initial treatment with 0.1 M nitric acid
containing 0.5% hydrogen peroxide (Reagent 1) is followed by
the addition of 0.25 M sodium hydroxide containing 7 mM of
the cationic surfactant cetyltrimethylammonium chloride (CTAC)
(Reagent 2).
In the current study, we examined the effect of different
surfactants in Reagent 2 on emission kinetics as well as total
light emitted from the acridinium ester labels and conjugates.
Specifically, light emission from each label or conjugate was
measured for a period of two minutes (in 0.5 s intervals) which was
sufficiently long for >90% emission under all conditions. Emission
kinetics, as reflected by the time required for >90% emission of
light from the labels and their conjugates, are tabulated in Tables 1
and 2 for the free labels 1a and 1b, the theophylline conjugates
2a and 2b as well the different protein conjugates of the labels 3a
and 3b. These emission times reflect the effect of the surfactant
in either accelerating or inhibiting the chemiluminescence rate of
the acridinium ester labels and conjugates. The total amount of
light emitted at the two minute measurement time, in the presence
of various surfactants, was used to calculate the effect of the
surfactant on light output (relative quantum yield). Total light
output in the absence of surfactant was normalized to a value of
one for all labels and conjugates. Relative light output values in
the presence of surfactants are listed in Tables 3 and 4 for the
free labels, the theophylline conjugates and the protein conjugates.
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 5092–5103 | 5095
©

View Article Online
Table 1 Time (secondsa) for emission of >90% total light of acridinium esters 1a and 1b and theophylline conjugates 2a and 2b in the presence of
different surfactants
Compound
No surfactant
CTAC
CTAOH
CTPAC
CTBAC
DDAO
DDAPS
SDS
Triton X-100
1a
1b
2a
2b
60.0
67.5
55.5
62.0
1.5
1.0
2.5
1.5
1.5
1.0
2.5
1.5
1.5
1.5
1.5
1.5
4.0
3.0
1.5
1.5
40.5
40.0
42.5
42.0
10.0
7.0
14.0
9.5
67.0
63.0
60.0
60.5
57.5
61.0
56.0
59.5
a Average of five replicates. Abbreviations used: CTAC = cetyltrimethylammonium chloride (cationic); CTAOH = cetyltrimethylammonium hy-
droxide (cationic); CTPAC = cetyltripropylammonium chloride (cationic); CTBAC = cetyltributylammonium chloride (cationic); DDAO = N,N-
dimethyldodecylamine oxide (zwitterionic); DDAPS = N,N-dimethyldodecylammonio-1,3-propane sulfonate (zwitterionic); SDS = sodium dodecyl
sulfate (anionic); Triton X-100 (non-ionic).
Table 2 Time (secondsa) for emission of >90% total light of acridinium ester–protein conjugates of labels 3a and 3b in the presence of different
surfactants
Conjugate
No surfactant
CTAC
CTAOH
CTPAC
CTBAC
DDAO
DDAPS
SDS
Triton X-100
Anti-TSH Mab-3a
Anti-TSH Mab-3b
Anti-HBsAg Mab-3a
Anti-HBsAg Mab-3b
Avidin-3a
64.5
61.5
64.5
60.5
89.5
74.5
2.5
1.5
3.0
2.0
3.5
1.0
3.0
3.0
3.0
3.0
3.0
1.5
3.0
2.0
5.0
3.0
21.0
2.5
3.5
2.5
5.5
4.0
9.0
2.0
46.5
40.0
46.0
39.0
72.5
53.0
14.5
9.5
13.5
9.0
61.0
29.0
89.0
86.5
90.5
88.0
87.5
80.0
62.5
59.5
61.0
58.5
89.0
73.5
Avidin-3b
a Average of five replicates.
Table 3 Observed enhancement of light output of acridinium esters 1a and 1b and theophylline conjugates 2a and 2b in the presence of different
surfactants at a measurement time of two minutes
Compound
No surfactant
CTAC
CTAOH
CTPAC
CTBAC
DDAO
DDAPS
SDS
Triton X-100
1a
1b
2a
2b
1
1
1
1
3.2
2.9
2.7
2.9
3.4
3.2
3.1
3.0
3.6
3.2
3.5
3.3
3.3
3.0
3.3
3.3
2.4
2.3
2.1
2.4
3.9
3.7
3.4
3.8
1.3
1.3
1.4
1.3
1.5
1.4
1.4
1.4
Table 4 Observed enhancement of light output of acridinium ester–protein conjugates of labels 3a and 3b in the presence of different surfactants at a
measurement time of two minutes
Conjugate
No surfactant
CTAC
CTAOH
CTPAC
CTBAC DDAO
DDAPS
SDS
Triton X-100
Anti-TSH Mab-3a
Anti-TSH Mab-3b
Anti-HBsAg Mab-3a
Anti-HBsAg Mab-3b
Avidin-3a
1
1
1
1
1
1
3.1
2.1
3.2
2.4
4.9
2.7
3.2
2.3
3.2
2.6
4.8
2.6
3.4
2.2
3.5
2.7
5.6
3.2
3.6
2.4
3.6
2.8
5.5
3.2
2.6
2.4
4.6
2.5
3.5
2.8
4.1
3.7
7.3
3.9
5.7
4.4
1.2
1.1
1.1
1.1
1.5
1.3
1.4
1.4
1.5
1.4
1.4
1.4
Avidin-3b
Thus, Tables 3 and 4 reflect the ability of the various surfactants
tested to enhance the total light emitted by the acridinium ester
labels and conjugates.
Among the different cationic surfactants, we examined the effect
of the counterion of cetyltrimethylammonium salts as represented
by the surfactants CTAOH (with hydroxide counterions) and
CTAC (with chloride counterions). Hydroxide ions are reported
to bind weakly to cetyltrimethylammonium micelles and this is
reflected in the increased CMC and ionization constant a of
CTAOH when compared to CTAC.22 The reactive ions in the
chemiluminescence reaction of acridinium esters are hydroperox-
ide ions which have been reported to display similar affinity to
cetyltrimethylammonium micelles as hydroxide ions.23 The effect
of head group size in cationic surfactants was also tested by
including the surfactants cetyltripropylammonium chloride (CT-
PAC) cetyltributylammonium chloride (CTBAC).24 Aggregates of
both these surfactants have been reported to bind anions less
strongly than cetyltrimethylammonium micelles owing to reduced
Chemiluminescence measurements were made using 10 femto-
moles (femtomole = 10-15 mole) of acridinium labels 1a and 1b and
theophylline conjugates 2a and 2b and 2.5 femtomoles of protein
conjugates in a total volume of 0.6 mL. Surfactant concentrations
in Reagent 2 were five times its reported CMC (critical micelle
concentration) in water. Final concentration of reagents was 0.25%
(~80 mM) peroxide in 0.125 M sodium hydroxide and the final
concentration of the surfactant was 2.5 times its reported CMC
in water. Thus, concentrations of peroxide and surfactant were in
vast excess compared to concentrations of the acridinium ester
labels and their conjugates. Details of these measurements can be
found in the experimental section.
5096 | Org. Biomol. Chem., 2011, 9, 5092–5103
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
polarity of their head groups which is reflected in lower CMC
values of these surfactants compared to CTAC.24 Two zwitteri-
onic surfactants, DDAO (N,N-dimethyldodecylamine oxide) and
DDAPS (N,N-dimethyldodecylammonio-1,3-propane sulfonate)
were included in the current study to examine their ef-
fects on acridinium ester chemiluminescence. While aggre-
gates of these surfactants are reported to attract anions only
weakly,25,26 they offer a non-polar environment similar to cationic
micelles.20,21 Finally, the effects of the anionic surfactant SDS
(sodium dodecyl sulfate) and the non-ionic surfactant tri-
ton X-100 on acridinium ester chemiluminescence were also
investigated.
Effect of surfactants on light emission kinetics
The effect of surfactants on emission kinetics of the two acridinium
ester labels and their conjugates is illustrated in Tables 1 and 2.
Light emission profiles for the labels 1a and 1b are illustrated in
Fig. 4 and 5, in the presence and absence of surfactants. Emission
profiles of the theophylline and protein conjugates were similar
and are illustrated in Fig. 6–13 of the ESI.† In the absence of
surfactant, light emission was quite slow for the labels and their
conjugates requiring a minute or more for >90% light emission.
In the presence of the cetyltrialkylammonium surfactants, CTAC,
CTAOH, CTPAC and CTBAC, light emission was observed to be
Fig. 4 Light emission profiles of acridinium ester label 1a in the absence and in the presence of various surfactants.
Fig. 5 Light emission profiles of acridinium ester label 1b in the absence and in the presence of various surfactants.
The Royal Society of Chemistry 2011 Org. Biomol. Chem., 2011, 9, 5092–5103 | 5097
This journal is
©

View Article Online
significantly faster and emission times were compressed to ~5 s
for the labels and their conjugates. The only exception was the
avidin conjugate of 3a which exhibited relatively slower emission
in the presence of CTPAC and CTBAC. Although aggregates of
CTPAC and CTBAC were expected to bind hydroperoxide ions
less strongly than micelles of the two cetyltrimethylammonium
surfactants, with the exception of the avidin conjugate of 3a, we did
not observe a significant attenuation in emission kinetics (Tables 1
and 2) because the surfactants were in vast excess. Protein pI in
general had little effect on the ability of the cationic surfactants in
accelerating emission kinetics. From the emission times in Tables 1
and 2, relative rate enhancements can be estimated by comparing
the emission times in the absence of surfactant and in the presence
of surfactant. These ratios indicate that the cationic surfactants,
especially CTAC and CTAOH, increase emission rates by at least
an order of magnitude for acridinium dimethylphenyl ester labels
and their conjugates.
Zwitterionic micelles are reported to attract anions weakly25,26
and both the zwitterionic surfactants, DDAO and DDAPS were
less effective than CTAC and CTAOH in enhancing the light emis-
sion kinetics of the labels and their conjugates. The amine oxide
surfactant, which has a more covalent head group, was observed
to be significantly less effective than the sulfobetaine surfactant in
enhancing light emission kinetics. Finally, no enhancement in light
emission kinetics could be observed in anionic micelles of sodium
dodecyl sulfate (SDS) as well as non-ionic surfactant triton X-100.
Cetyltrimethylammonium hydroperoxide (CTAOOH) has been
shown to catalyze phosphate ester hydrolysis by concentrating
the reactive components in the micellar phase thereby leading
to faster observed rates.23,27,28 Our observations on the effects of
aggregates of cetyltrimethylammonium salts on the light emission
kinetics of the acridinium dimethylphenyl ester labels 1a and 1b
as well as their conjugates are consistent with these reports. The
reaction of hydroperoxide ions with these acridinium esters and
their conjugates appears to be a slow step (conversion of 2 to
3 in Fig. 2) in the chemiluminescence reaction pathway that is
catalyzed by cetyltrimethylammonium micelles, resulting from (a)
binding of these labels and their conjugates to the aggregates and,
(b) increased local concentration of hydroperoxide ions at the
positively charged micellar surface. Attenuation of this positive
charge as exemplified by the two zwitterionic surfactants decreases
the local concentration of hydroperoxide ions leading to slower
light emission. Aggregates of the anionic surfactant SDS, as well as
the non-ionic surfactant triton X-100 are not expected to concen-
trate negatively charged hydroperoxide ions at their surfaces and
consequently both these surfactants did not accelerate emission
kinetics. Comparison of the emission profiles of 1a and 1b (Fig. 4
and 5) indicate that initial rates decrease in the order CTAC
(cationic) ~ CTAOH (cationic) ~ CTPAC (cationic) ~ CTBAC
(cationic) > DDAPS (zwitterionic) > DDAO (zwitterionic) >
triton X 100 (non-ionic) ~ no surfactant > SDS (anionic). Similar
conclusions can be drawn from comparing the emission profiles
of the theophylline conjugates 2a and 2b (Fig. 6 and 7, ESI†) and
the antibody conjugates of 3a and 3b (Fig. 8–13, ESI†).
ester labels and their conjugates as illustrated in Tables 3 and 4.
In the present study, the total amount of light was measured for
two minutes for each label and conjugate both in the absence of
surfactant and the presence of various surfactants. The relative
quantum yields of the labels 1a and 1b and their conjugates were
assigned a value of one in the absence of surfactant to facilitate
interpretation of surfactant effects on total light output. Light
output on different proteins was similar for the two labels.
As illustrated in Tables 3 and 4, aggregates of the cetyltrimethy-
lammonium surfactants, which were the most effective in enhanc-
ing light emission kinetics, were observed to enhance light output
approximately 3-fold from the labels and theophylline conjugates
although more variation was observed for the protein conjugates.
Chemiluminescence enhancement of the labels 1a and 1b, the two
theophylline conjugates 2a and 2b and the protein conjugates of 3a
and 3b was also slightly greater in the presence of the two cationic
surfactants CTPAC and CTBAC with larger head groups.
Micelles of the two zwitterionic surfactants DDAO and DDAPS
were observed to be less effective than cationic cetyltrimethy-
lammonium surfactants in enhancing emission kinetics but both
surfactants enhanced light output from the two labels as well their
conjugates. The magnitude of this enhancement in DDAO was
slightly lower compared to the cationic surfactants. However, the
sulfobetaine surfactant DDAPS was more effective than CTAC
and CTAOH in enhancing light output from the two labels and
their conjugates as illustrated in Tables 3 and 4.
Micelles of the non-ionic surfactant triton X-100, which did
not enhance emission kinetics of the acridinium ester labels and
their conjugates, were also only marginally effective in enhancing
light output. Similarly, the anionic surfactant SDS showed only
marginal enhancement of light output for the labels 1a and 1b and
their conjugates.
What is the mechanism by which cationic and zwitterionic
surfactants enhance chemiluminescence of acridinium esters 1a
and 1b (Fig. 1) and their conjugates? When considering plausible
mechanisms, the impact of the micellar environment on all the
reaction steps outlined in Fig. 2 must be assessed. Clearly, as
noted earlier, formation of the hydroperoxide adduct 3 (Fig. 2)
from the acridinium ester 2 is accelerated by cationic surfactants
which is manifested as faster light emission. Formation of excited
state acridone 6, which is the light emitting species, is postulated
to occur either from dioxetane 4 or dioxetanone 5 (Fig. 2) by the
CIEEL mechanism9–11 and the micellar environment may influence
not only the formation of all three chemical species but may also
affect emission from excited state acridone 6.
Chemiluminescence from the decomposition of dioxetanes via
the CIEEL mechanism results in the formation of singlet excited
states of the primary emitters in high quantum yields.9,11,12,29
Chemiluminescence emission spectra of acridinium esters are iden-
tical to the fluorescence spectra of the corresponding acridones
which display mono-exponential fluorescence decay reflecting
emission from a single excited state.10b Chemiluminescence emis-
sion spectra of acridinium ester conjugates of fluorescent dyes,
resulting from very efficient energy transfer from excited state
acridone to the fluorescent moieties, are also identical to the
fluorescent spectra of the dyes.30
Effect of surfactants on light output
Published studies31 on the fluorescence of acridones in various
media are thus useful in understanding the impact of the micellar
environment on emission from excited state acridone 6 (Fig. 2).
In addition to emission kinetics, surfactants were also observed to
exert significant effects on the total light emitted by the acridinium
5098 | Org. Biomol. Chem., 2011, 9, 5092–5103
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
Hinze et al. have reported that the fluorescence quantum yields,
fluorescence lifetimes and fluorescence emission spectra of N-
methylacridone (NMA) in water, as well as in solutions of cationic,
zwitterionic, anionic and non-ionic surfactants are very similar.31a
The fluorescence quantum yield of this acridone was close to unity
in these different media as well as in alcoholic solvents (methanol,
ethanol and 2-propanol) but decreased in less polar solvents such
as dimethyl sulfoxide and dimethyl formamide. For example, the
emission wavelength maximum, fluorescence quantum yield and
fluorescence lifetime for NMA in water was observed to be 431 nm,
1.00 and 15.8 ns, respectively. In a 5 mM solution of CTAC, the
emission wavelength maximum, fluorescence quantum yield and
fluorescence lifetime were 427 nm, 0.97 and 15.2 ns, respectively. In
a previous study, we had also observed that the chemiluminescence
emission wavelength maximum of an N-sulfopropyl acridinium
ester, which forms electronically excited N-sulfopropylacridone,
in the presence of CTAC is 426 nm.2
Siegmund et al. studied singlet–triplet dynamics of NMA in
a wide range of solvents31b and also observed lower fluorescence
quantum yields in non-polar solvents for NMA as well as for
acridone and N-phenylacridone.31b,c In halogenated solvents where
the fluorescence quantum yield of NMA was significantly lower,
kinetics of intersystem crossing were 5–10 fold higher.31b A related
study by Mory et al. on a series of acridones with different N-alkyl
groups showed that the first singlet excited state is populated in
polar solvents and the first triplet state is populated in non-polar
solvents.31d
bromide based on the magnitude of the observed catalysis which
was slightly greater for DDAPS. Catalysis was only very modest
in the presence of non-ionic surfactants presumably because of
increased hydration of these surfactant aggregates.
Formation of both dioxetane 4, from intramolecular cyclization
of hydroperoxide adduct 3 as well as dioxetanone 5 from elimina-
tion of the phenol from 4 (Fig. 2), also involve dispersal of negative
charge in the transition states and are expected to be facilitated
by reduced medium polarity. Either of these intermediates may
be the immediate precursor to excited state acridone 68–10 and
therefore, an increase in their yields should result in a concomitant
increase in the yield of excited state acridone. Consistent with
this expectation, we have observed that both cationic micelles of
cetyltrialkylammonium salts as well as zwitterionic micelles of
DDAPS enhance the chemiluminescence of acridinium esters and
their conjugates but significant chemiluminescence enhancement
was not observed in aggregates of the non-ionic surfactant triton
X-100 because of increased hydration of the latter. Chemilumines-
cence enhancement was maximal for the zwitterionic surfactant
DDAPS as well as the two cationic surfactants CTPAC and
CTBAC with large head groups indicating that medium-polarity is
an important factor affecting formation of excited state acridone
6. In the unimolecular decarboxylation of 6-nitrobenzisoxazole-
3-carboxylate, little catalysis was noted in the presence of anionic
SDS micelles.33 Similarly, we observed minimal enhancement
in chemiluminescence of acridininium dimethyl esters and their
conjugates in the presence of this anionic surfactant.
The fact that the fluorescence quantum yield of NMA is
the same in water and surfactant solutions31a along with data
published by Siegmund et al.31b on singlet–triplet dynamics of
acridones suggests that in the chemiluminescent reaction of
acridinium ester, (a) singlet–triplet dynamics of excited state
acridone 6 are not influenced by surfactants and, (b) solubilization
of excited state acridone 6 occurs in a relatively polar region
of the micelle, i.e., the Stern layer of the micellar phase which
is considered to be ‘alcohol-like’ in polarity.19 Thus, published
studies strongly suggest that surfactants can only influence the
chemiluminescence reaction steps of acridinium esters preceding
the formation of excited state acridone 6 but not its emission.
To understand how surfactant aggregates might affect forma-
tion of dioxetane 4 and dioxetanone 5, it is useful to examine
published reports on their impact on analogous unimolecular
reactions. Unimolecular reactions such as the decarboxylation of
6-nitrobenzisoxazole-3-carboxylate,19 the intramolecular cycliza-
tion reactions of ortho-haloalkyl-substituted phenoxides19 and 1,2-
elimination reactions20 all involve dispersal of negative charge in
the transition states, and are catalyzed by aggregates of cationic
and zwitterionic surfactants because of reduced polarity (alcohol-
like) at the micellar phase. These observations are consistent with
classical studies by Hughes, Ingold and co-workers on the effects
of solvent polarity on organic reactions.32 In the intramolecular
cyclization reactions of ortho-haloalkyl-substituted phenoxides,
from a comparison of the cyclization rates, relative rate enhance-
ment in CTAC aggregates was observed to be similar to that
observed in ethanol whereas rate enhancement in CTPAC and
CTBAC micelles, with more hydrophobic surfactant head groups,
was greater and similar to that observed in 2-propanol.20d It was
postulated that zwitterionic micelles derived from DDAPS offered
a less polar environment than micelles of cetyltrimethylammonium
Finally, it is also important to examine the potential impact of
the micellar environment on the CIEEL mechanism leading to ex-
cited state acridone 6 (Fig. 2). Details of the reaction steps involved
in the conversion of dioxetane 4 or dioxetanone 5 to excited state
acridone 6 are presently unclear,10 and there is also controversy re-
garding the efficacy of an intermolecular versus an intramolecular
electron transfer process on the chemiluminescence of dioxetanes.
For example, in the triggered luminescence of spiroadamantyl-
substituted dioxetanes, Adam et al. observed an increase in the
excitation yield of the light emitting species with increased solvent
viscosity.34 These observations were attributed to the ‘solvent-
cage’ effect on the CIEEL mechanism of this dioxetane and were
considered to support the intermolecular back-electron transfer
(BET) mechanism as opposed to an intramolecular process leading
directly to the excited state phenolate anion. However, Ciscato
et al.35 recently reported that an intermolecular BET reaction is
a low yield process compared to an intramolecular BET reaction
for acridinium-dioxetanes. The latter study is more relevant to
our work and since surfactants enhance the light emission of
acridinium esters, therefore it is unlikely that they would promote
an intermolecular BET reaction for decomposition of dioxetane 4
or dioxetanone 5 (Fig. 2).
Conclusions
We have examined the effects of surfactants on light emission
from two hydrophilic acridinium dimethylphenyl ester labels
that are currently used in automated immunoassays for clinical
diagnostics. Our results indicate that surfactants influence the
chemiluminescence reaction pathway (Fig. 2) at two different steps
of the overall process. The initial reaction of hydroperoxide ions
with the acridinium ester is accelerated by cationic surfactants
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 5092–5103 | 5099
©

View Article Online
leading to faster light emission. Surfactants also enhance total
light output by facilitating formation of the dioxetane 4 and/or
the dioxetanone5 if the latter is indeed atrue reaction intermediate.
Literature evidence31 strongly suggests that surfactants do not
affect the fluorescence quantum yield, the fluorescence lifetime,
emission spectrum or singlet–triplet dynamics of excited state
acridone, which is the light emitting species.
From a practical point of view, cationic surfactants such as
CTAC, that are effective in both enhancing light output and
accelerating emission kinetics of acridinium dimethylphenyl esters
are the most useful for automated instruments where a fast read
time is required to maintain high throughput.
filtered and concentrated under reduced pressure to afford a
viscous oil that solidified upon storage to a waxy solid. HPLC
analysis of the product was performed using a Phenomenex, 10
micron, C18 3.9 mm ¥ 25 cm column and a 30 min gradient of 10 →
70% B (A = water with 0.05% TFA, B = MeCN with 0.05% TFA;
TFA = trifluoroacetic acid) at a flow rate of 1.0 mL min-1 and UV
detection at 260 nm. Product was observed eluting at 21.6 min as
a broad peak. Yield = 3.2 g (quantitative); dH (500 MHz, CDCl3)
1.59–1.70 (m, 4 H), 2.16 (s, 3 H), 2.37 (t, 4 H, J = 6.5), 3.24 (q,
4 H, J = 6.0), 5.07 (s, 4 H), 5.55 (br s, 2 H), 7.27–7.40 (m, 10 H);
dC (125 MHz, CDCl3) 26.77, 39.84, 41.66, 55.81, 66.45, 127.98,
128.06, 128.44, 136.71, 155.45; MALDI-TOF MS m/z 414.4 (M
+ H)+; HRMS m/z 414.2385 (M + H)+ (414.2393 calculated).
Experimental
(b) N,N-Bis(3-benzyloxycarbamoylpropyl)methylammonium-
1,3-propane sulfonate, compound iii. A solution of N,N-bis(3-
benzyloxycarbamoylpropyl)methylamine (1.2 g, 2.9 mmol) in
anhydrous DMF (15 mL) was treated with 1,3-propane sultone
(0.71 g, 5.8 mmol, 2 equivalents). The reaction was heated at
145 ◦C under nitrogen for 1 h. HPLC analysis of a small portion
of the reaction mixture was performed using a Phenomenex, 10
micron, C18 3.9 mm ¥ 25 cm column and a 30 min gradient of 10
→ 70% B (A = water with 0.05% TFA, B = MeCN with 0.05%
TFA; TFA = trifluoroacetic acid) at a flow rate of 1.0 mL min-1
and UV detection at 260 nm. Product was observed eluting at
19.6 min (~80% conversion) with starting material eluting at
21.6 min. The reaction mixture was concentrated under reduced
pressure and the recovered oil was dissolved in methanol (20 mL).
TLC analysis on silica using 40% methanol, 60% ethyl acetate
indicated clean separation of product (Rf ª 0.2) from starting
material (Rf ª 0.3). The above reaction was repeated on the
same scale and the combined reaction mixture was purified by
flash chromatography on silica using 40% methanol, 60% ethyl
acetate as eluent. Yield = 1.55 g (60%); white foam; dH (500 MHz,
CD3OD) 1.87–1.96 (m, 4 H), 2.06–2.14 (m, 2H), 2.84 (t, 2 H,
J = 6.5), 2.99 (s, 3 H), 3.21 (t, 4 H, J = 6.2), 3.24–3.30 (m, 4 H),
3.44–3.50 (m, 2 H), 5.08 (s, 4 H), 7.27–7.33 (m, 2 H), 7.35 (br d, 8
H); dC (125 MHz, CF3COOD) 17.65, 20.18, 37.54, 47.18, 48.20,
59.34, 60.02, 70.81, 128.15, 128.40, 129.07, 132.45; MALDI-TOF
MS m/z 536.4 (M + H)+; HRMS m/z 536.2435 (M + H)+
(536.2430 calculated).
General
Chemicals and surfactants were purchased from Sigma–Aldrich
(Milwaukee, Wisconsin, USA) unless indicated otherwise. N,N-
Bis(3-aminopropyl)methyl amine was purchased from TCI Amer-
ica. The syntheses of the acridinium compounds 1a and 3a, and
the theophylline conjugate 2a have been described previously.5
Cetyltripropylammonium chloride (CTPAC) and cetyltributylam-
monium chloride (CTBAC) were synthesized using a literature
procedure.24
All final acridinium esters and theophylline conjugates were
analyzed and purified by HPLC using a Beckman-Coulter
HPLC system. MALDI-TOF (Matrix-Assisted Laser Desorption
Ionization-Time of Flight) mass spectrometry was performed
using a Voyager DETM BiospectrometryTM Workstation from
Perkin–Elmer. This is a benchtop instrument operating in the
linear mode with a 1.2 meter ion path length, flight tube. Spectra
were acquired in positive ion mode. For acridinium esters and
theophylline conjugates, a-cyano-4-hydroxycinnamic acid was
used as the matrix and spectra were acquired with an accelerating
voltage of 20 000 volts and a delay time of 100 ns. For protein
conjugates, sinapinic acid was used as the matrix and spectra were
acquired with an accelerating voltage of 25 000 volts and a delay
time of 85 ns.
For HRMS (High Resolution Mass Spectra), samples were
dissolved in HPLC-grade methanol and analyzed by direct-flow
injection (injection volume = 5 mL) ElectroSpray Ionization (ESI)
on a Waters Qtof API US instrument in the positive ion mode.
Optimized conditions were as follows: Capillary = 3000 kV, Cone =
35, Source T = 120 ◦C, Desolvation T = 350 ◦C. NMR spectra
were recorded on a Varian 500 MHz spectrometer.
(c) N,N-Bis(3-aminopropyl)methylammonium-1,3-propane sul-
fonate, compound iv. N,N-Bis(3-benzyloxycarbamoylpropyl)-
methylammonium-1,3-propane sulfonate (0.8 g, 1.49 mmol) was
stirred in 15 mL of 33% HBr/AcOH at room temperature for
24 h. Ether (100 mL) was then added and a white, granular solid
separated out. The product was allowed to settle and the ether was
decanted. This process was repeated twice with ether (2 ¥ 50 mL).
Finally, the product was dried under vacuum. The recovered
viscous oil was dissolved in 5–6 mL water, frozen at -80 ◦C
and lyophilized to dryness to afford a glassy solid. TLC analysis
on silica using 25% ammonia, 75% methanol and ninhydrin for
visualization showed a single spot of Rf ª 0.2.
Yield = 0.766 g (quantitative); dH (500 MHz, CF3COOD) 2.77–
2.83 (m, 6H), 3.45 (s, 3H), 3.59 (br t, 2H), 3.70 (br s, 4H), 3.96
(br s, 6H), 7.42 (br s, 6H); dC (125 MHz, CF3COOD) 18.15, 20.73,
38.03, 47.78, 48.76, 59.49, 61.30; MALDI-TOF MS m/z 268.2
(M + H)+; HRMS m/z 268.1686 (M + H)+ (268.1695 calculated).
Synthesis of compounds 1b, 2b and 3b (Fig. 3)
(a) N,N-Bis(3-benzyloxycarbamoylpropyl)methylamine, com-
pound ii. A solution of N,N-bis(3-aminopropyl)methyl amine
(1 g, 6.9 mmol) in chloroform (40 mL) was treated with N-
(benzyloxycarbonyloxy)succinimide (3.78 g, 15.2 mmol, 2.2 equiv-
alents). The reaction was stirred at room temperature for 16 h. TLC
analysis of the reaction mixture on silica using 15% methanol
in ethyl acetate showed the formation of a polar product in a
clean reaction. The reaction mixture was diluted with chloroform
(40 mL) and washed with saturated aqueous sodium bicarbonate
solution. It was then dried over anhydrous magnesium sulfate,
5100 | Org. Biomol. Chem., 2011, 9, 5092–5103
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
(d) Compound 1b.
carboxylphenyl
A
solution of 2¢,6¢-dimethyl-4¢-
fractions containing product 3b were combined, frozen at -80 ◦C
and lyophilized to dryness to a yellow, fluffy powder. Yield =
28.7 mg (50%); dH (500 MHz, CF3COOD) 2.27 (br m, 4 H), 2.44
(br s, 2 H), 2.62 (s, 8 H), 2.71 (br s, 2 H), 2.87 (br s, 2 H), 3.03
(br s, 2 H), 3.13 (s, 4 H), 3.26 (s, 3 H), 3.50 (br s, 2 H), 3.64 (m,
6 H), 3.83 (br s, 4 H), 3.91 (br s, 2 H), 5.94 (br t, 2 H), 7.78 (s, 2
H), 8.21 (t, 2 H), 8.63 (t, 2 H), 8.82 (d, 2 H, J = 8.6), 9.00 (d, 2
H, J = 8.9); dC (125 MHz, CF3COOD) 16.3, 17.7, 19.8, 20.1, 22.1,
23.1, 24.5, 24.9, 29.1, 31.9, 33.8, 37.4, 47.5, 48.2, 50.1, 60.0, 60.2,
118.3, 123.4, 124.9, 127.9, 128.5, 129.4, 130.6, 131.9, 140.4, 141.5,
148.7, 151.5, 168.8, 173.9, 176.7, 177.5, 180.2; MALDI-TOF MS
m/z 955.2 (M + H)+; HRMS m/z 954.3274 (M + H)+ (954.3265
calculated).
N-sulfopropylacridinium-9-carboxylate2,5
(30 mg, 0.061 mmole) in DMF (3 mL) was treated with diiso-
propylethylamine (0.016 mL, 0.0917 mmole, 1.5 equivalents)
and N,N,N¢,N¢-tetramethyl-O-(N-succinimidyl)uronium tetraflu-
oroborate (TSTU) (22 mg, 0.0732 mmol, 1.2 equivalents). The
reaction was stirred at room temperature. After 15 min, HPLC
analysis of a small portion of the reaction mixture was performed
using a Phenomenex, 10 micron, C18 3.9 mm ¥ 25 cm column and
a 30 min gradient of 10 → 70% B (A = water with 0.05% TFA,
B = MeCN with 0.05% TFA) at a flow rate of 1.0 mL min-1 and
UV detection at 260 nm. Product NHS ester (compound v) was
observed eluting at 20 min and was the major component. This
DMF solution of the NHS ester, compound v, was added dropwise
to a solution of N,N-bis(3-aminopropyl)methylammonium-1,3-
propane sulfonate, compound iv, (0.136 g, 0.0304 mmol, 5
equivalents, HBr salt) dissolved in DMF (1 mL) and 0.25 M
sodium bicarbonate (1 mL). The reaction was stirred at room
temperature. After 3 h, HPLC analysis showed clean conversion
to the product 1b, eluting at 12.4 min. Using a 40 min gradient of
10 → 40% B (A = water with 0.05% TFA, B = MeCN with 0.05%
TFA), the product eluted at 19.2 min. The product was purified
by preparative HPLC using a YMC, 10 micron, C18 30 ¥ 250 mm
column and 40 min gradient of 10 → 40% B (A = water with
0.05% TFA, B = MeCN with 0.05% TFA) at a solvent flow rate of
20 mL min-1 and UV detection at 260 nm. The HPLC fractions
containing product 1b were combined and concentrated under
reduced pressure to yield a yellow, sticky solid. Yield = 45 mg
(86%, TFA salt); dH (500 MHz, CF3COOD) 2.40 (br s, 2 H), 2.56
(br s, 10 H), 2.95 (br s, 2 H), 3.24 (s, 3 H), 3.46 (br t, 4 H), 3.65
(br s, 4 H), 3.72–3.86 (br m, 6 H), 5.87 (br t, 2 H), 7.71 (s, 2 H),
8.15 (t, 2 H), 8.57 (t, 2 H), 8.76 (d, 2 H, J = 8.6), 8.92 (d, 2 H, J =
9.1); dC (125 MHz, CF3COOD) 16.3, 17.7, 20.2, 22.2, 23.2, 37.4,
37.8, 47.4, 48.1, 48.2, 50.0, 59.1, 60.1, 60.9, 118.3, 123.4, 124.9,
127.9, 128.4, 129.5, 130.6, 131.9, 140.5, 141.5, 148.8, 151.5, 171.8;
MALDI-TOF MS m/z 743.2 (M + H)+; HRMS m/z 743.2786
(M + H)+ (743.2784 calculated).
(f) Theophylline conjugate 2b.
A
solution of com-
pound 1b (22 mg, 0.0257 mmol, TFA salt) in DMSO
(2 mL) was added to 8-carboxypropyltheophylline (5.5 mg,
0.0207 mmol, Sigma) followed by diisopropylethylamine
(0.0088 mL, 0.0505 mmol, 2 equivalents) and (benzotriazol-
1-yl-oxy)tris(dimethylamino)phosphonium hexafluorophosphate
(BOP) (13.7 mg, 0.031 mmol, 1.2 equivalents). The reaction was
stirred at room temperature for 16 h and then analyzed by HPLC
using a Phenomenex, 10 micron, C18 3.9 mm ¥ 25 cm column
and a 40 min gradient of 10 → 40% B (A = water with 0.05%
TFA, B = MeCN with 0.05% TFA) at a flow rate of 1.0 mL min-1
and UV detection at 260 nm. Product was observed eluting at
23.6 min and was the major component. The product was purified
by preparative HPLC using a YMC, 10 micron, C18 30 ¥ 250 mm
column and 40 min gradient of 10 → 40% B (A = water with
0.05% TFA, B = MeCN with 0.05% TFA) at a solvent flow
rate of 20 mL min-1 and UV detection at 260 nm. The HPLC
fractions containing product 2b were combined, frozen at -80 ◦C
and lyophilized to dryness to a yellow, fluffy powder. Yield = 7.5 mg
(37%); dH (500 MHz, CF3COOD) 2.36 (br s, 2 H), 2.50 (br s, 4 H),
2.62 (br s, 2 H), 2.67 (s, 6 H), 2.79 (br s, 2 H), 3.06 (m, 2 H), 3.30
(s, 3 H), 3.46 (t, 2 H, J = 7.8), 3.51 (t, 2 H, J = 7.2), 3.65 (br s, 4
H), 3.70 (s, 3 H), 3.81–3.89 (m, 4 H), 3.91 (s, 3 H), 3.94 (t, 2 H, J =
6.4), 5.99 (m, 2 H), 7.82 (s, 2 H), 8.25 (t, 2 H), 8.67 (t, 2 H), 8.87 (d,
2 H, J = 8.6), 9.04 (d, 2 H, J = 9.5); MALDI-TOF MS m/z 992.0
(M + H)+; HRMS m/z 991.3680 (M + H)+ (991.3694 calculated).
(e) Compound 3b. A solution of compound 1b (45 mg,
0.0525 mmol) in methanol (3.6 mL) and water (0.4 mL) was
treated with diisopropylethylamine (0.053 mL, 0.303 mmol, 5.8
equivalents) and glutaric anhydride (34.5 mg, 0.303 mmol, 5.8
equivalents). The reaction was stirred at room temperature. After
15 min, HPLC analysis of a small portion of the reaction mixture
was performed using a Phenomenex, 10 micron, C18 3.9 mm ¥
25 cm column and a 30 min gradient of 10 → 70% B (A = water
with 0.05% TFA, B = MeCN with 0.05% TFA) at a flow rate of
1.0 mL min-1 and UV detection at 260 nm. The glutarate derivative
was observed eluting at 14 min and was the major component. The
solvent was then removed under reduced pressure. The residue
was dissolved in DMF (3.6 mL) and water (0.4 mL). This solution
was treated with diisopropylethylamine (0.106 mL, 10 equivalents)
and TSTU (182 mg, 10 equivalents). The reaction was stirred at
room temperature. After 10 min, HPLC analysis showed complete
conversion to the product 3b eluting at 15.3 min. The product was
purified by preparative HPLC using a YMC, 10 micron, C18 30 ¥
250 mm column and 40 min gradient of 10 → 40% B (A = water
with 0.05% TFA, B = MeCN with 0.05% TFA) at a solvent flow
rate of 20 mL min-1 and UV detection at 260 nm. The HPLC
General procedure for protein labeling with acridinium esters
Three proteins were used for labeling with the acridinium ester
labels 3a and 3b; a murine anti-TSH monoclonal antibody with
an acidic pI = 5.6 (TSH = Thyroid Stimulating Hormone), a murine
anti-HBsAg monoclonal antibody with a neutral pI = 7 (HBsAg =
Hepatitis B Surface Antigen) and egg white avidin, pI = 10.5. The
following is a typical procedure for labeling the three proteins with
10 equivalents input of acridinium ester label.
The anti-TSH murine monoclonal antibody (1 mg, 6.67
nanomoles, stock solution 5 mg mL-1, 0.2 mL) was diluted with
0.2 mL of 0.1 M sodium carbonate, pH 9. The protein solution
was treated with DMSO solutions of acridinium esters as follows:
for labeling with 10 equivalents of 3a, 0.0129 mL of a 5 mg mL-1
DMSO solution of the compound was added and, for labeling
with 10 equivalents of compound 3b, 0.0127 mL of a 5 mg mL-1
D◦MF solution was added. The labeling reactions were stirred at
4 C for 16 h and were then transferred to 4 mL AmiconTM filters
(MW 30 000 cutoff) and diluted with 3.5 mL de-ionized water.
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 5092–5103 | 5101
©

View Article Online
Table 5 Acridinium ester label incorporation in protein conjugates
with reactions with the various surfactants. The output from the
luminometer instrument was expressed as R.L.U.s (Relative Light
Units). For evaluation of surfactants, the surfactant was either
omitted or replaced in Reagent 2 with one of the following
surfactants at five times its reported critical micelle concentration
(CMC) in water.
Observed Observed Iincrease # of
Protein/conjugate
mass
in mass
labels
Unlabeled anti-TSH Mab
Anti-TSH Mab-3a
Anti-TSH Mab-3b
Unlabeled anti-HBsAg Mab
Anti-HBsAg Mab-3a
Anti-HBsAg Mab-3b
Unlabeled avidin
151336
155238
155898
149947
153834
154167
63928
—
—
3902
4562
—
3887
4220
—
4.6
5.4
—
4.6
5.0
—
(a) Cetyltrimethylammonium chloride (CTAC), CMC
1.4 mM;22,24
=
(b) Cetyltrimethylammonium hydroxide (CTAOH), CMC =
2.3–3.4 mM;22
Avidin-3a
Avidin-3b
68024
68516
4096
4588
4.8
5.5
(c) Cetyltripropylammonium chloride (CTPAC), CMC =
0.65 mM24
(d) Cetyltributylammonium chloride (CTBAC), CMC
=
=
0.52 mM,24
The volume was reduced to ~0.1 mL by centrifuging at 4000g
for 10 min. The concentrated conjugate solutions were diluted
with 4 mL de-ionized water and centrifuged again to reduce the
volume. This process was repeated a total of four times. Finally,
the concentrated conjugates were diluted with 0.1 mL de-ionized
water. The conjugates were analyzed by MALDI-TOF mass
spectrometry, using the Voyager-DE instrument from Perkin–
Elmer, to measure acridinium compound incorporation. Typically,
this entailed measuring the molecular weight of the unlabeled
protein and the labeled protein. The acridinium compound
label contributed the observed difference in mass of these two
measurements. By knowing the molecular weight of the specific
acridinium compound label, the extent of label incorporation of
that specific acridinium compound could thus be calculated. Label
incorporation in each protein is tabulated in Table 5.
(e) N,N-Dimethyldodecylamine oxide (DDAO), CMC
2 mM;25
(f) N,N-Dimethyldodecylammonio-1,3-propane sulfonate
(DDAPS), CMC = 3.17 mM;36
(g) Sodium dodecyl sulfate (SDS), CMC = 8 mM;37
(h) Triton X-100, CMC = 0.24 mM.38
Acknowledgements
We thank Mary Foltz for pI measurements and Drs. Qingping
Jiang and Alan Burkhardt for helpful suggestions with the
manuscript.
References
1 L. J. Kricka, Anal. Chim. Acta, 2003, 500, 279–286.
2 A. Natrajan, D. Sharpe, J. Costello and Q. Jiang, Anal. Biochem., 2010,
406, 204–213.
3 S.-J. Law, T. Miller, U. Piran, C. Klukas, S. Chang and J. Unger,
J. Biolumin. Chemilumin., 1989, 4, 88–98.
4 S.-J. Law, C. S. Leventis, A. Natrajan, Q. Jiang, P. B. Connolly, J. P.
Kilroy, C. R. McCudden and S. M. Tirrell, US Pat. 5,656,426, 1997.
5 (a) A. Natrajan, D. Sharpe and Q. Jiang, US Pat. 6,664,043 B2, 2003;
(b) A. Natrajan and D. Wen, Green Chem., 2011, 13, 913–921.
6 I. Weeks, I. Behesti, F. McCapra, A. K. Campbell and J. S. Woodhead,
Clin. Chem., 1983, 29/8, 1474–1479.
7 S. C. S. Chang and T. E. Miller, US Pat. 4,927,769, 1990.
8 F. McCapra, Acc. Chem. Res., 1976, 9/6, 201–208.
9 F. McCapra, D. Watmore, F. Sumun, A. Patel, I. Beheshti, K.
Ramakrishnan and J. Branson, J. Biolumin. Chemilumin., 1989, 4, 51–
58.
10 (a) J. Rak, P. Skurski and J. Błaz˙ejowski, J. Org. Chem., 1999, 64,
3002–3008; (b) K. Krzymin´ski, A. Oz˙o´g, P. Malecha, A. D. Roshal, A.
Wro´blewska, B. Zadykowicz and J. Błaz˙ejowski, J. Org. Chem., 2011,
76, 1072–1085; (c) K. Krzymin´ski, A. D. Roshal, B. Zadykowicz, A.
Białk-Bielin´ska and A. Sieradzan, J. Phys. Chem. A, 2010, 114, 10550–
10562.
11 (a) F. McCapra, J. Chem. Soc., Chem. Commun., 1977, 946–948; (b) F.
McCapra, I. Beheshti, A. Burford, R. A. Hann and K. A. Zaklika,
J. Chem. Soc., Chem. Commun., 1977, 944–946.
12 G. B. Schuster, Acc. Chem. Res., 1979, 12, 366–373.
13 J. Lasovsky´, M. Rypka and J. Slouka, J. Lumin., 1995, 65, 25–32.
14 N. Dan, M. L. Lau and M. L. Grayeski, Anal. Chem., 1991, 63, 1766–
1771.
15 P. J. Sheridan, US Pat. 5,853,974, 1998.
16 F. J. Bagazgoitia, J. L. Garcˆıa, C. Die´quez, I. Weeks and J. S. Woodhead,
J. Biolumin. Chemilumin., 1988, 2, 121–128.
17 (a) J. Ladd, Z. Zhang, S. Chen, J. C. Hower and S. Jiang, Biomacro-
molecules, 2008, 9, 1357–1361; (b) Y. Chang, S. Chen, Z. Zhang and S.
Jiang, Langmuir, 2006, 22, 2222–2226; (c) Z. Zhang, T. Chao, S. Chen
and S. Jiang, Langmuir, 2006, 22, 10072–10077; (d) W. K. Cho, B. Kong
and I. S. Choi, Langmuir, 2007, 23, 5678–5682; (e) G. Cheng, Z. Zhang,
S. Chen, J. D. Bryers and S. Jiang, Biomaterials, 2007, 28, 4192–4199;
Light emission
Chemiluminescence of acridinium esters was measured on an
Autolumat LB953 Plus luminometer from Berthold Technologies.
Acridinium esters and their conjugates were diluted for chemi-
luminescence measurements in an aqueous buffer of 10 mM
disodium hydrogen phosphate, 0.15 M NaCl, 8 mM sodium azide,
0.015 mM BSA, pH 8.0. Protein conjugates were serially diluted
to a final concentration of 25 pM based on protein concentration
measured using the BCA Protein Assay from Pierce. Acridinium
ester labels 1a and 1b and theophylline conjugates 2a and 2b were
likewise diluted to a final concentration of 100 pM. A 0.010 mL
volume of each diluted acridinium ester sample was dispensed
into the bottom of a cuvette, representing the chemiluminescence
from 2.5 femtomoles of acridinium ester-labeled protein conjugate
and 10 femtomoles of acridinium ester labels 1a and 1b and
theophylline conjugates 2a and 2b. Cuvettes were placed into the
primed LB953 and the chemiluminescence reaction was initiated
with the sequential addition of 0.3 mL of Reagent 1, a solution
of 0.5% hydrogen peroxide in 0.1 M nitric acid followed by the
addition of 0.3 mL of Reagent 2, a solution of 0.25 M sodium
hydroxide containing surfactant at 5 times its reported critical
micelle concentration (CMC) in water. Each chemiluminescence
flash curve was measured in 240 intervals of 0.5 s (2 min total time)
from the point of chemiluminescence initiation with the addition
of surfactant solution in 0.25 M NaOH. Each chemiluminescence
reaction was carried out five times, averaged and converted to a
percentage of the chemiluminescence accumulated up to each time
interval. Chemiluminescence values for 2 min collection times were
also normalized for comparison to reactions without surfactant
5102 | Org. Biomol. Chem., 2011, 9, 5092–5103
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
(f) H. Kitano, A. Kawasaki, H. Kawasaki and S. Morokoshi, J. Colloid
Interface Sci., 2005, 282, 340–348.
28 C. A. Bunton and H. J. Foroudian, Langmuir, 1993, 9, 2832–2835.
29 (a) C. Lee and L. A. Singer, J. Am. Chem. Soc., 1980, 102, 3823–3829;
(b) A. P. Schaap, T.-S. Chen, R. S. Handley, R. DeSilva and B. P. Giri,
Tetrahedron Lett., 1987, 11, 1155–1158.
30 Q. Jiang, J. Xi, A. Natrajan, D. Sharpe, M. Baumann, R. Hilfiker , E.
Schmidt, P. Senn, F. Thommen, A. Waldner, A. Alder and S.-J. Law,
US Pat. 6,165,800, 2000.
31 (a) W. L. Hinze, T. E. Riehl and H. N. Singh, Anal. Chem., 1984, 56,
2180–2191; (b) M. Siegmund and J. Bendig, Ber. Bunsenge. Phys. Chem.,
1978, 82, 1061–1068; (c) M. Siegmund and J. Bendig, Z. Naturforsch,
1980, 35a, 1076–1086; (d) S. Mory, H.-J. Weigman, A. Rosenfeld, M.
Siegmund, R. Mitzner and J. Bendig, Chem. Phys. Lett., 1985, 115,
201–204.
32 (a) E. D. Hughes and C. K. Ingold, J. Chem. Soc., 1935, 244–255;
(b) E. D. Hughes, Trans. Faraday Soc., 1941, 37, 603–631; (c) E. D.
Hughes and C. K. Ingold, Trans. Faraday Soc., 1941, 37, 657–685;
(d) K. A. Cooper, M. L. Dhar, E. D. Hughes, C. K. Ingold, B. J.
MacNulty and L. I. Woolf, J. Chem. Soc., 1948, 2043–2049.
33 C. A. Bunton and M. J. Minch, Tetrahedron Lett., 1970, 44, 3881–3884.
34 W. Adam, I. Bronstein, A. V. Trofimov and R. F. Vasil’ev, J. Am. Chem.
Soc., 1999, 121, 958–961.
18 A. T. Marttila, K. J. Airenne, O. H. Laitinen, T. Kulik, E. A. Bayer, M.
Wilchek and M. S. Kulomaa, FEBS Lett., 1998, 441, 313–317.
19 (a) C. A. Bunton, F. Nome, F. H. Quina and L. S. Romsted, Acc. Chem.
Res., 1991, 24, 357–364; (b) P. Scrimin, P. Tecilla, U. Tonellato and C. A.
Bunton, Colloids Surf., A, 1998, 144, 71–79; (c) L. S. Romsted, C. A.
Bunton and J. Yao, Curr. Opin. Colloid Interface Sci., 1997, 2, 622–628.
20 (a) P. D. Profio, R. Germani, G. Savelli, G. Cerichelli, N. Spreti and
C. A. Bunton, J. Chem. Soc., Perkin Trans. 2, 1996, 1505–1509; (b) G.
Cerichelli, L. Luchetti, G. Mancini, M. N. Muzzioli, R. Germani, P. P.
Ponti, N. Spreti, G. Savelli and C. A. Bunton, J. Chem. Soc., Perkin
Trans. 2, 1989, 1081–1085; (c) G. Cerichelli, G. Mancini, L. Luchetti,
G. Savelli and C. A. Bunton, J. Phys. Org. Chem., 1991, 4, 71–76;
(d) G. Cerichelli, L. Luchetti, G. Mancini, G. Savelli and C. A. Bunton,
Langmuir, 1996, 12, 2348–2352.
21 (a) L. Brinchi, P. D. Profio, R. Germani, G. Savelli and C. A. Bunton,
Langmuir, 1997, 13, 4583–4587; (b) L. Brinchi, R. Germani, G. Savelli,
N. Spreti, R. Ruzziconi and C. A. Bunton, Langmuir, 1998, 14, 2656–
2662; (c) L. Brinchi, R. Germani, G. Savelli and C. A. Bunton, J. Phys.
Org. Chem., 1999, 12, 890–894.
22 L. Sepu´lveda and J. Corte´s, J. Phys. Chem., 1985, 89, 5322–5324.
23 J. Toullec and M. Moukawim, Chem. Commun., 1996, 221–222.
24 R. Bacaloglu, C. A. Bunton and F. Ortega, J. Phys. Chem., 1989, 93,
1497–1502.
25 A. Natrajan and C. N. Sukenik, J. Org. Chem., 1988, 53, 3559–3563.
26 C. A. Bunton, M. M. Mhala and J. R. Moffat, J. Org. Chem., 1987, 52,
3832–3835.
35 Luiz Francisco, M. L. Ciscato, F. H. Bartoloni, D. Weiss, R. Beckert
and W. J. Baader, J. Org. Chem., 2010, 75, 6574–6580.
36 Y. Wang, X. Huang, Y. Li, J. Wang and Y. Wang, Colloids Surf., A,
2009, 333, 108–114.
37 P. Horowitz, J. Colloid Interface Sci., 1977, 61, 197–198.
38 (a) S. Ross and J. P. Olivier, J. Phys. Chem., 1959, 63, 1671–1674; (b) G.
Valde´s-D´ıaz, S. Rodr´ıguez-Calvo, A. Pe´rez-Gramatges, M. Rapado-
Paneque, F. A. Fernandez-Lima, C. R. Ponciano and E. F. da Silveira,
J. Colloid Interface Sci., 2007, 311, 253–261.
27 V. K. Balakrishnan, E. Buncel and G. W. Vanloon, Environ. Sci.
Technol., 2005, 39, 5824–5830.
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 5092–5103 | 5103
©