Effect of branching in remote substituents on light emission and stability of chemiluminescent acridinium esters

Article abstract of DOI:10.1039/c4ra02516d

Acridinium dimethylphenyl esters are widely used as chemiluminescent labels in automated immunoassays for clinical diagnostics in Siemens Healthcare Diagnostics' ADVIA Centaur systems. Light emission from these labels and their conjugates is triggered with alkaline peroxide. Excited state acridone is believed to be the light emitting species that is formed from the initial peroxide adduct which subsequently undergoes a series of reactions leading to scission of the phenolic ester bond. Dioxetane and/or dioxetanone intermediates have been proposed as immediate precursors of excited state acridone. Despite the fact that acridinium esters have been widely used as chemiluminescent labels for decades, a substantive theoretical framework to guide acridinium ester design with improved properties over the basic structure is unavailable. We have relied on a more empirical approach to devise new acridinium esters with improved stability, higher light yield, fast light emission, low non-specific binding and improved immunoassay performance. In the current study, we have investigated the effect of branching in remote alkoxy substituents attached to C-2 and C-7 of the acridinium ring on light emission and chemiluminescence stability of two acridinium esters. We selected two, high light output acridinium dimethylphenyl esters that we described previously as a basis for these studies and report the synthesis of two new C-2 and C-7 alkoxy-substituted labels, compounds 5 and 10, with improved chemiluminescence stability and faster light emission respectively. Compound 5 exhibited better long term stability at both pH 6 and 7.4 (≥10%) compared to its unbranched counterpart compound 11 whereas compound 10 with branched hexa(ethylene) glycol substituents exhibited >4-fold faster light emission compared to its unbranched counterpart 12. Both parameters are important for immunoassay performance in automated instruments. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4ra02516d

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Effect of branching in remote substituents on light
emission and stability of chemiluminescent
acridinium esters†
Cite this: RSC Adv., 2014, 4, 21852
Received 21st March 2014
Accepted 7th May 2014
*
Anand Natrajan and David Wen
DOI: 10.1039/c4ra02516d
www.rsc.org/advances
Acridinium dimethylphenyl esters are widely used as chemilumi-
nescent labels in automated immunoassays for clinical diagnostics
in Siemens Healthcare Diagnostics' ADVIA Centaur® systems.
Light emission from these labels and their conjugates is triggered
with alkaline peroxide. Excited state acridone is believed to be the
light emitting species that is formed from the initial peroxide
Introduction
Acridinium dimethylphenyl esters1 (structures illustrated in
Fig. 1) are widely used as chemiluminescent labels in auto-
mated immunoassays for clinical diagnostics in Siemens
Healthcare Diagnostics' ADVIA Centaur® systems. Light emis-
sion from these labels is triggered by the sequential addition of
two reagents. An initial treatment with 0.1 M nitric acid con-
taining 0.5% hydrogen peroxide converts the non-chemilumi-
nescent water adducts of the acridinium esters, (I in Fig. 2) that
are also commonly referred to as pseudobases,2 to the chemi-
luminescent acridinium forms of the labels (II in Fig. 2).
Subsequent addition of 0.25 M sodium hydroxide ionizes the
hydrogen peroxide which reacts with the acridinium ester
eventually leading to excited state acridone VI. Dioxetane IV and
dioxetanone V have been proposed as intermediates in the
chemiluminescent reaction pathway of acridinium esters.3
Recent theoretical studies suggest that excited state acridone VI
is formed directly from dioxetane III.3 Details regarding this
process are not well understood although light emission is
believed to result from a single electronically excited state of the
acridone.3d,e
adduct which subsequently undergoes
a series of reactions
leading to scission of the phenolic ester bond. Dioxetane and/or
dioxetanone intermediates have been proposed as immediate
precursors of excited state acridone. Despite the fact that acri-
dinium esters have been widely used as chemiluminescent labels
for decades, a substantive theoretical framework to guide acri-
dinium ester design with improved properties over the basic
structure is unavailable. We have relied on a more empirical
approach to devise new acridinium esters with improved stability,
higher light yield, fast light emission, low non-specific binding and
improved immunoassay performance. In the current study, we
have investigated the effect of branching in remote alkoxy
substituents attached to C-2 and C-7 of the acridinium ring on
light emission and chemiluminescence stability of two acridinium
esters. We selected two, high light output acridinium dimethyl-
phenyl esters that we described previously as a basis for these
studies and report the synthesis of two new C-2 and C-7 alkoxy-
substituted labels, compounds 5 and 10, with improved chem-
iluminescence stability and faster light emission respectively.
Compound 5 exhibited better long term stability at both pH 6
and 7.4 ($10%) compared to its unbranched counterpart
compound 11 whereas compound 10 with branched hexa-
(ethylene) glycol substituents exhibited >4-fold faster light emis-
sion compared to its unbranched counterpart 12. Both parameters
are important for immunoassay performance in automated
instruments.
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
Fig. 1 Structures of chemiluminescent acridinium dimethylphenyl
ester labels1 that are used in automated immunoassays in Siemens
Healthcare Diagnostics' ADVIA Centaur® systems.
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c4ra02516d
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Fig. 2 Simplified reaction pathway of chemiluminescence from N-sulfopropyl acridinium esters. In the ADVIA Centaur® system, light emission is
triggered by the sequential addition of 0.1 M nitric acid containing 0.5% hydrogen peroxide followed by 0.25 M sodium hydroxide containing the
cationic surfactant cetyltrimethylammonium chloride (CTAC). Acid treatment converts the water adduct I to the acridinium ester II which then
reacts with alkaline peroxide ultimately resulting in excited state acridone VI. Dioxetane IV and/or dioxetanone V are proposed reaction inter-
mediates. CTAC compresses emission times and increases light yield.
The cationic surfactant cetyltrimethylammonium chloride immunoassay performance.1 These studies have also provided
(CTAC) is included in 0.25 M sodium hydroxide when light us with important insights into the chemiluminescence
emission is triggered from acridinium dimethylphenyl esters pathway of acridinium esters as well as some useful principles
and their conjugates. In the absence of CTAC, light emission for acridinium ester design.
from the labels and conjugates is relatively slow and can take up
In a recent study,1a,b we evaluated the effect of methoxy
to a minute for complete emission. Overall light yield in the groups placed at various positions in the acridinium ring (see
absence of CTAC is also signicantly lower. Inclusion of CTAC Fig. 1 for numbering system) on light emission from the cor-
compresses emission times of the labels to <5 seconds and also responding acridinium esters. Maximum light yield was
increases overall light yield 3–4 fold.1c,e These effects result from observed with the placement of two methoxy groups at C-2 and
the high local concentration of hydroperoxide ions at the C-7 of the acridinium ring. Although
a 2,7-dimethoxy-
cationic micellar surface which accelerates their reaction with substituted acridinium dimethylphenyl ester proved to be
micelle-bound acridinium ester, as well as a less polar micellar considerably less stable than an unsubstituted acridinium
medium which in turn facilitates formation of dioxetane and ester, replacement of the methoxy groups with bulkier
dioxetanone precursors of the primary emitter, excited state methoxyhexa(ethylene) glycol groups surprisingly improved
acridone VI. The observed enhancement in acridinium ester chemiluminescence stability without compromising the high
chemiluminescence (light yield) is quite sensitive to the polarity light yield observed with the methoxy-substituted analog.1b An
of the micellar interface.1c
acridinium ester label with C-2 and C-7, methoxyhexa(ethylene)
Although acridinium esters have been used as chemilumi- glycol groups along with a hexa(ethylene) glycol linker attached
nescent labels for quite some time now, but for a few mecha- to the phenol, also exhibited low non-specic binding and
nistic studies,3c–e a theoretical framework which can be used to enhanced assay sensitivity in a sandwich assay for TSH (TSH ¼
predict and tailor the properties of these commercially-useful thyroid stimulating hormone).1b A structural variant of this
labels is not available. In its absence, we have relied on a more label1a with a simpler linker attached to the phenol to facilitate
empirical approach and in recent publications, we have out- protein labeling (Fig. 3, compound 12), is currently used for
lined studies that have proved useful to devise new acridinium measurement of the cardiac marker troponin I peptide (TnI) in
ester structures with improved stability, higher light yield, fast the ADVIA Centaur® TnI-Ultra assay.
light emission, low non-specic binding and improved
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Fig. 3 Structures of C-2 and C-7 dialkoxy-substituted acridinium dimethylphenyl esters. Compound 11 (ref. 1f) is a strongly anionic N-sulfo-
propyl acridinium ester with two additional sulfonates appended to C-2 and C-7 of the acridinium ring. Compound 12 (ref. 1a) is also an N-
sulfopropyl acridinium ester with two methoxy hexa(ethylene) glycol groups attached to C-2 and C-7 of the acridinium ring. Compounds 5 and
10 are acridinium esters synthesized in the current study. Compound 5 is an analog of 11 with additional branching at the phenolic ethers at C-2
and C-7. Compound 10 is an analog of 12 with branched hexa(ethylene) glycol groups attached to C-2 and C-7 of the acridinium ring.
Instability of acridinium esters is generally attributed to
The synthesis of the anionic label 5 is illustrated in Fig. 4 and
hydrolysis of the phenolic ester bond.4 The improved stability of follows an analogous route that we described recently for
compound 12 compared to the methoxy-substituted analog compound 11.1f In the case of 11, 2,7-dihdroxyacridine ester 1
clearly illustrates that structural changes in alkoxy substituents was alkylated at the two phenolic hydroxyl groups as well as the
that increase their steric bulk even when located remote from acridine nitrogen in one step with 1,3-propane sultone.1f For the
the phenolic ester linkage can affect stability of acridinium O-alkylation reaction leading to compound 5, we used 1,3-
esters. Because branching is expected to further increase the butane sultone 2 as the alkylating reagent. The sultone 2 was
steric bulk of substituents, in the current study, we have synthesized from the methane sulfonate (mesylate) diester of
investigated the effect of branching at these remote alkoxy 1,2-propane diol using a literature procedure.5 As before, the
substituents on the light emission and chemiluminescence O-alkylation reaction was conducted in the ionic liquid 1-butyl-
stability of two acridinium dimethylphenyl esters (Fig. 3).
3-methylimidazolium hexauorophosphate [BMIM][PF6] in the
presence of potassium carbonate as base. Despite the lower
reactivity of 2 which entails ring opening at a secondary carbon
instead of a primary carbon in 1,3-propane sultone, neverthe-
less, in preparative reactions, we observed excellent conversion
to the O-dialkylated product 3 when the crown ether dibenzo-18-
crown-6 was included in the alkylation reaction even with only 3
equivalents of the sultone 2. Interestingly, the lowered reactivity
of 2 ensured no N-alkylation at the acridine nitrogen which is
much more sterically hindered6 even when 15 equivalents of
sultone 2 were used in the O-alkylation reaction. The sultone 2
is chiral and two sets of diastereomers are possible for the
O-alkylated product 3. HPLC analysis of the O-alkylation reac-
tion using a high resolution column did indeed indicate the
formation of a pair of diastereomers eluting close together
(Fig. S1a and b, ESI†). A small quantity of each isomer was
isolated by HPLC (Fig. S1c and f, ESI†) and both showed very
similar high eld proton NMR spectra (Fig. S1d and g, ESI†) and
Results and discussion
1. Synthesis of acridinium esters and bovine serum albumin
(BSA) conjugates
To investigate the effect of branching in remote C-2 and C-7
alkoxy substituents in acridinium esters on their properties, we
selected two different acridinium esters 5 and 10 for synthesis
(Fig. 3). Compound 5 is an analog of 11, a strongly anionic label
whose synthesis we described recently.1f Compound 5 contains
additional branching at the phenolic ethers at C-2 and C-7. The
introduction of two methyl groups in 5 represents only a small
structural change in the acridinium ring compared to 11 and is
not expected to signicantly increase the hydrophobicity of the
acridinium ester. Compound 10 is an analog of 12 with
branched instead of linear hexa(ethylene) glycol groups
attached to C-2 and C-7 of the acridinium ring.
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Fig. 4 Synthetic scheme for compound 5. Reagents: (a) dibenzo-18-crown-6, potassium carbonate, [BMIM][PF6]; (b) 1,3-propane sultone, 2,6-
di-tert-butylpyridine, [BMIM][PF6]; (c) 2 M HCl; (d) N,N,N0,N0-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU), diisopropyle-
thylamine (DIPEA), DMF.
identical mass spectra (Experimental section). Separation of the further reacted with glutaric anhydride followed by activation of
isomers was not practical for preparative purposes and there- the glutarate intermediate to give target 10. Although compound
fore, they were carried forward together to the next step in an 10 contains a different linker compared to compound 12, we
one pot reaction which entailed O-alkylation as described above reasoned that the slightly longer linker in 10 would facilitate
followed by N-alkylation of the acridine nitrogen with 1,3- protein labeling and offset the steric hindrance caused by the
propane sultone in [BMIM][PF6] in the presence of 2,6-di-tert- bulky ether substituents at C-2 and C-7 in 10. This proved to be
butylpyridine as base.6a The N-sulfopropyl acridinium carboxy- true during labeling of BSA with ve equivalents input of each of
lic acid 4 was obtained aer hydrolysis of the N-sulfopropyl the various acridinium esters as can be noted in Table 1. Mass
acridinium methyl ester and was subsequently converted to the spectral analysis indicated the incorporation of 3–5 labels for the
target compound 5 using standard chemistry for N-hydroxy- various labels using the labeling protocol described in the
succinimide (NHS) ester formation.
experimental section. HPLC chromatograms of all synthetic
Synthesis of the acridinium ester 10 with branched hexa- intermediates and nal compounds of Fig. 4 and 5 are shown in
(ethylene) glycol groups at C-2 and C-7 of the acridinium ring also the ESI (Fig. S1–S7).†
started with 2,7-dihydroxyacridine ester 1 as illustrated in Fig. 5.
Compound
6 which, is a branched isomer of methoxy-
2. Chemiluminescence measurements, stability and
pseudobase formation
hexa(ethylene) glycol, was synthesized using a literature proce-
dure and followed a common synthetic protocol that is used for
the assembly of these types of branched glycerol derivatives.7
Thus, as described in the published procedure, reaction of
epichlorohydrin with methanol afforded 1,3-dimethoxyglycerol8a,b
which was again treated with epichlorohydrin in the presence of
base to give compound 6 (ref. 8b) (Experimental section).
Coupling of compound 6 and dihydroxyacridine ester 1 to estab-
lish the phenolic ether linkages was accomplished using the
Mitsunobu reaction9 which gave the C-2 and C-7 di-substituted
acridine ester 7. Acridine ester 7 was then N-alkylated with 1,3-
propane sultone in [BMIM][PF6] in the presence of 2,6-di-tert-
butylpyridine to introduce the N-sulfopropyl group. Hydrolysis of
the N-sulfopropyl acridinium methyl ester intermediate gave the
acridinium carboxylic acid 8. Activation of the carboxylic acid 8
followed by condensation with a commercially available linker
2,20-(ethylenedioxy)bis(ethylamine) gave compound 9 which was
Light yield (specic activity) and emission times from BSA
conjugates of the acridinium esters 5, 10 and their counterparts
11 and 12 (without branching at C-2 and C-7) are listed in Table
1. The emission proles are illustrated in Fig. 6 and 7. For all the
acridinium esters, we used BSA conjugates instead of the free
labels to ensure complete solubility of the labels in aqueous
buffer especially at high dilution that were needed for
measurements in the linear range of the photomultiplier tube
in the luminometer. (In our previous study we observed that
micellar effects on the chemiluminescence of acridinium ester
labels are very similar for the free labels as well as the conju-
gates of the labels.1c,e) The conjugates ꢀ2 mg mLꢁ1 were serially
diluted into buffer to nal concentrations of approximately 0.3
nanomolar (nM) and chemiluminescence was measured using a
luminometer from Berthold Technologies. Light emission
from 0.01 mL samples were initiated by the sequential addition
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Fig. 5 Synthetic scheme for compound 10. Reagents: (a) triphenylphosphine (TPP), diisopropylazodicarboxylate (DIAD), triethylamine (TEA),
tetrahydrofuran; (b) 1,3-propane sultone, 2,6-di-tert-butylpyridine, [BMIM][PF6]; (c) 2 M HCl; (d) TSTU, diisopropylethylamine DIPEA, DMF; (e)
2,20-(ethylenedioxy)bis(ethylamine), DMF; (f) glutaric anhydride, DIPEA, DMF; (g) TSTU, DIPEA, DMF.
critical micelle concentration10) of the cationic surfactant CTAC.
Light was measured for 20 seconds integrated at 0.1 s intervals.
Table 1 Acridinium ester incorporation in BSA conjugates measured
my mass spectroscopy, specific chemiluminescent activity (SCA) of the
labels and time for $95% emission. Light emission was triggered by the
For measurements in the absence of CTAC, light was measured
sequential addition of 0.3 mL reagent 1 (0.1 M nitric acid + 0.5%
peroxide) followed by 0.3 mL reagent 2 (7 mM CTAC in 0.25 M NaOH).
for 2 minutes integrated at 0.5 s intervals. (Additional details
can be found in the Experimental section.) The output of the
instrument is expressed as Relative Light Units (RLUs).
(RLU ¼ Relative Light Unit). The strongly anionic labels 5 and 11 as well
as the labels 10 with branched hexa(ethylene) glycol groups, and 12
with linear hexa(ethylene) glycol groups respectively all showed the
As can be noted from Table 1, the specic chemilumines-
same light yield because of the same C-2 and C-7 dialkoxy substitution
cent activity (SCA), expressed as RLUs per mole, of the various
pattern. Acridinium ester 10 showed the fastest emission time
labels are quite comparable. This is unsurprising because all
four acridinium esters 5, 10, 11 and 12 have the same C-2 and
No. of labels by
mass spectroscopy
SCA ꢂ 10ꢁ19
Time for $95%
emission (seconds)
Label
RLUs per mole
C-7 dialkoxy-substituted motif which is the main determinant
of light yield based on our earlier study.1a,b Emission times, as
reected by the time needed for $95% emission of total light,
however showed a signicant difference for the two acridi-
nium esters 10 and 12 containing linear versus branched hexa-
(ethylene) glycol substituents. Acridinium ester 10 with
branched hexa(ethylene) glycol groups showed much faster
emission (>4-fold faster) compared to 12 for $95% emission of
total light in the presence of CTAC (Table 1). A comparison of
the emission proles of these acridinium esters in the
5
4
5
4
3
6.1
4.6
4.3
5.3
3.8
1.2
3.0
5.3
10
11
12
of 0.3 mL of 0.1 M nitric acid with 0.5% hydrogen peroxide
(reagent 1) followed by the addition of 0.3 ml of sodium
hydroxide (reagent 2) with and without 7 mM (5 times the
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Fig. 6 Kinetics of light emission of BSA conjugates of acridinium esters 5, 10, 11 and 12 in the presence of CTAC. Emission kinetics of the strongly
anionic labels 5 and 11 were quite similar. On the other hand, emission from acridinium ester 10 with branched hexa(ethylene) glycol groups at
C-2 and C-7 was rapid when compared to compound 12 with linear hexa(ethylene) glycol groups are C-2 and C-7 of the acridinium ring.
presence of CTAC (Fig. 6) and in its absence (Fig. 7) showed CTAC, light emission from 10 and 12 was relatively slow
that the differences in the emissive rates of these two (Fig. 7) but 10 still showed comparatively faster emission
compounds is an intrinsic one based on structural differences than 12.
between the C-2 and C-7 alkoxy substituents. In the absence of
Fig. 7 Kinetics of light emission of BSA conjugates of acridinium esters 5, 10, 11 and 12 in the absence of CTAC. Emission kinetics of the strongly
anionic labels 5 and 11 were again quite similar to each other in the absence of surfactant. Emission from acridinium ester 10 with branched
hexa(ethylene) glycol groups at C-2 and C-7 was faster when compared to compound 12 with linear hexa(ethylene) glycol groups are C-2 and
C-7 of the acridinium ring.
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On the other hand, for the two strongly anionic labels 5 and and perhaps insignicant difference in stability between the
11, emission times were quite similar to each other both in the two structures at either pH (Table 3).
presence and absence of CTAC as can be noted from Table 1 and
Chemiluminescence instability of acridinium esters is
the emission proles in Fig. 6 and 7. In this case, branching at attributed to hydrolysis of the phenolic ester with simple
the phenolic ethers at C-2 and C-7 had little impact on emission unsubstituted phenyl esters exhibiting poor stability.4 This
times. As noted for 10 and 12, in the absence of CTAC light instability is alleviated considerably for acridinium dimethyl-
emission from 5 and 11 was relatively slow but was much faster phenyl esters due to steric shielding afforded by the two methyl
in the presence of CTAC.
groups anking the phenolic ester.4 Another factor we have
We next compared the chemiluminescent stability of the BSA observed that impacts chemiluminescence stability of acridi-
conjugates of the acridinium esters 5, 10, 11 and 12 at pH 6 and nium esters is the pKa of the acridinium–pseudobase tran-
7.4 over a period of ve weeks and the results are shown in sition.1b, f The acridinium form I (Fig. 1) is expected to be more
Tables 2 and 3. The conjugates of the acridinium esters were susceptible to hydrolysis of the phenolic ester due to electron
diluted into phosphate buffer at pH 6 and 7.4 as described withdrawal from the quaternary acridinium nitrogen. Thus,
earlier and were stored at room temperature (25ꢃ C) protected acridinium esters with a higher pKa for the acridinium to
from light. Chemiluminescence from 0.01 mL samples was pseudobase transition are expected to be less stable and this
measured periodically (ve replicates) and the observed RLUs prediction appears to be generally true.1b,f (At a given pH, a
were averaged and converted to percentages. The initial greater proportion of the acridinium ester is in the labile acri-
observed RLUs at the rst time point (day 1) was assigned a dinium form if it has a higher pKa.) In fact pseudobase-type
value of 100% and residual chemiluminescence as a percentage adducts formed by the addition of various sulfyhydryl-contain-
of this initial value reects the chemiluminescence stability. ing nucleophiles have been shown to enhance the hydrolytic
(Details of the measurements can be found in the Experimental stability of simple acridinium phenyl esters.2c
section.) As observed previously for an antibody conjugate,1f the
Acridinium esters with a higher pKa for pseudobase forma-
BSA conjugate of the anionic label 11 in the current study, was tion are also predicted to have a less electrophilic C-9 because
again observed to be more stable at acidic pH 6 than at pH 7.4 pseudobase formation occurs at a higher pH. Does this trans-
(Table 2). Interestingly, while branching at the phenolic ethers late to a slower reaction with hydroperoxide ions which add at
in the structurally analogous, strongly anionic label 5 had a C-9 as a rst step in the chemiluminescence reaction pathway of
minimal impact on emission kinetics, its chemiluminescence acridinium esters (Fig. 2)? Our empirical observations1f suggest
stability was observed to be better at both pH 6 and 7.4. that pKa of pseudobase formation of different acridinium esters
Consistent with our previous observations for 11, the conjugate are not necessarily correlated with observed emission kinetics.
of 5 was also observed to be more stable at pH 6 compared to pH Thus, in our previous study,1f we noted that acridinium esters
7.4. Stability comparison of the BSA conjugates of compounds with different pKas for pseudobase formation exhibited similar
10 and 12 with branched and linear hexa(ethylene) glycol emission kinetics.
groups respectively, on the other hand, showed a much smaller
Table 2 Residual chemiluminescence (%, average of five replicates) of BSA conjugates of the strongly anionic acridinium esters 5 and 11 as a
function of pH at room temperature. Branching at the phenolic ethers in 5 improved long term stability at both pH 6 and pH 7.4. (CV ¼ coefficient
of variation of measurements)
Day
11, pH ¼ 6 (%CV)
5, pH ¼ 6 (%CV)
11, pH ¼ 7.4 (%CV)
5, pH ¼ 7.4 (%CV)
1
7
14
21
28
35
100 (2.5)
93 (2.3)
77 (3.8)
69 (3.1)
63 (3.9)
59 (3.2)
100 (2.7)
92 (4.6)
82 (3.3)
77 (2.8)
72 (3.2)
69 (2.6)
100 (2.2)
84 (2.7)
68 (2.5)
59 (4.2)
54 (4.1)
49 (2.2)
100 (3.8)
85 (2.4)
79 (4.4)
73 (4.7)
68 (1.0)
62 (2.2)
Table 3 Residual chemiluminescence (%, average of five replicates) of BSA conjugates of acridinium esters 10 and 12 as a function of pH at room
temperature. Both acridinium esters with linear or branched hexa(ethylene) glycol groups at C-2 and C-7 exhibited similar stability. (CV ¼
coefficient of variation of measurements)
Day
12, pH ¼ 6 (%CV)
10, pH ¼ 6 (%CV)
12, pH ¼ 7.4 (%CV)
10, pH ¼ 7.4 (%CV)
1
7
14
21
28
35
100 (4.9)
85 (3.8)
70 (3.3)
64 (3.3)
59 (3.2)
57 (1.5)
100 (5.8)
89 (7.7)
82 (4.3)
68 (3.6)
62 (4.3)
59 (5.7)
100 (7.2)
85 (3.5)
70 (2.6)
61 (3.5)
55 (3.4)
50 (4.4)
100 (2.0)
88 (3.3)
75 (4.4)
69 (5.9)
59 (3.6)
55 (3.7)
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Fig. 8 pH titrations of compounds 10 and 12 illustrating conversion of the acridinium form II to the pseudobase I (refer to Fig. 1). The branched
hexa(ethylene) glycol groups in 10 induced a small shift in the pKa for pseudobase formation to acidic pH.
Fig. 9 pH titrations of compounds 11 and 5 illustrating conversion of the acridinium form II to the pseudobase I. Branching at the phenolic ethers
in 5 induced a small shift in the pKa for pseudobase formation to acidic pH.
Are the observed differences in chemiluminescence stability the conjugation of the acridinium ring and results in a loss of
of the acridinium esters 5 and 10 with branched C-2 and C-7 the long wavelength absorption band. Typically, a sharp tran-
phenolic ethers versus their unbranched counterparts 11 and 12 sition over a narrow range of pH is noted for the acridinium to
due to a shi in the pKa for pseudobase formation to acidic pH? pseudobase formation as illustrated in Fig. 8 and 9. As can be
To answer this question, we measured the pKa of pseudobase noted from Fig. 8, introduction of branching at the phenolic
formation (conversion of II to I in Fig. 2) of the various acridi- ethers in 5 induced a small shi in the pKa (ꢀ0.5 unit) for
nium esters using a simple pH titration method that we pseudobase formation to acidic pH when compared to 11 which
described previously1e,f (Fig. 8 and 9). Briey, the long wave- would be more conducive to the improved chemiluminescence
length absorption band of the acridinium chromophore is stability of 5 observed in Table 2. However, stability differences
measured as a function of pH. Pseudobase formation disrupts between 10 and 12 were minimal as noted in Table 3 yet 10 with
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branched hexa(ethylene) glycol groups also showed a small and [BMIM][PF6] was dried under high vacuum over P2O5 prior
equivalent shi in pKa for pseudobase formation to acidic pH to use.
when compared to its linear counterpart 12. The reason why
All nal acridinium esters and intermediates were analyzed
branching at a remote substituent in an acridinium ester affects and/or puried by HPLC using a Beckman-Coulter HPLC
its pKa is unclear but may reect relief of steric crowding caused system. Flash chromatography was performed using an ‘Auto-
by a at acridinium ring and branched functional groups ash’ system from Teledyne Isco. MALDI-TOF (Matrix-Assisted
attached in close proximity to the acridinium ring system. Laser Desorption Ionization-Time of Flight) mass spectroscopy
Pseudobase formation presumably relieves this steric crowding was performed using a Voyager De Biospectrometry Worksta-
because the acridine ring in the pseudobase is no longer at. tion from ABI. This is a bench top instrument operating in the
Observed differences in chemiluminescence stability of the linear mode with a 1.2 meter ion path length, ight tube.
various acridinium esters, 5 versus 11 and 10 versus 12 however Spectra were acquired in positive ion mode. For small mole-
cannot be attributed entirely to shis in the pKa for pseudobase cules, a-cyano-4-hydroxycinnamic acid was used as the matrix
formation to acidic pH at least for 10 and 12. These remote and spectra were acquired with an accelerating voltage of 20 000
substituents must also affect formation and/or decomposition volts and a delay time of 100 ns. For protein conjugates, sina-
of the sterically-crowded tetrahedral intermediates involved in pinic acid was used as the matrix and spectra were acquired
phenolic ester hydrolysis.
with an accelerating voltage of 25 000 volts and a delay time of
Finally, what explains the difference in emission kinetics 85 ns.
between 10 and 12? If addition of hydroperoxide ions at C-9 is
For HRMS (High Resolution Mass Spectra), samples were
equally facile for 10 and 12 (Fig. 2), then the increase in emissive dissolved in HPLC-grade methanol and analyzed by direct-ow
rates for 10 must result from faster decomposition of the injection (injection volume ¼ 5 mL) ElectroSpray Ionization (ESI)
dioxetane intermediate. If the steric bulk of remote C-2 and C-7 on a Waters Qtof API US instrument in the positive ion mode.
functional groups can affect stability of the phenolic ester as we Optimized conditions were as follows: capillary ¼ 3000 kV, cone
observed previously1b and in the current study, it is reasonable ¼ 35, source temperature ¼ 120 ꢃC, desolvation temperature ¼
to expect that the steric bulk of remote substituents may also 350 ꢃC. NMR spectra were recorded on a Varian 500 MHz
affect the stability and decomposition of the dioxetane inter- spectrometer. IR spectra of neat samples were recorded on a
mediate III (Fig. 2). The fast light emission observed for 10 Bruker TENSOR37 FT-IR spectrometer, ATR mode on ZnSe
compared to 12 suggests that the branched hexa(ethylene) crystal. UV-visible spectra were recorded on a Beckman DU 7500
glycol group imposes greater steric constraints than the linear spectrophotometer. Chemiluminescence measurements were
hexa(ethylene) glycol substituent which can adopt an extended carried out using a Berthold Technologies' AutoLumat Plus
conformation in aqueous solution.
LB953 luminometer.
2. Synthesis of acridinium esters (Fig. 4, 5, and S1–S7 ESI†)
Conclusions
Compound 3. A mixture of compound 1 (10 mg, 0.024
mmol), potassium carbonate (10 mg, 0.072 mmol), dibenzo-18-
crown-6 (26 mg, 0.072 mmol) and 1,3-butane sultone 2 (49 mg,
In the current study we have described the syntheses of two new
acridinium esters 5 and 10 based on the high light yielding, C-2
and C-7 dialkoxy-substituted structural motif. The new struc-
tures contain additional branching at these alkoxy groups
which were observed to impact both chemiluminescence
stability and emission kinetics despite being located remote
from the reaction centers affecting these two parameters. From
a practical point of view, both new structures 5 and 10 are
improvements over their corresponding predecessors 11 and
12. In the case of the strongly anionic label 5, better chem-
iluminescence stability was observed compared to 11 which is
useful for reagent stability. In the case of 10, the improvement
in chemiluminescence stability was not signicant compared to
12 but the new label showed much faster light emission which
is another useful feature in automated immunoassays for
fast throughput because of reduced cycle times needed for
each assay.
0.359
mmol)
in
1-butyl-3-methylimidazolium
hexa-
uorophosphate [BMIM][PF6] (0.5 mL) was heated at 155 ꢃC for
2 hours. The reaction was diluted with ethyl acetate (5 mL) and
applied to a silica column equilibrated with ethyl acetate. The
column was rst eluted with ethyl acetate (525 mL) to elute
unreacted starting material and dibenzo-18-crown-6 followed by
40% methanol in ethyl (500 mL) to elute product. The product
fractions were combined and concentrated under reduced
pressure. The product was analyzed by analytical HPLC using a
Phenomenex, Kinetex C18, 50 ꢂ 4.6 mm, 2.6 micron column
with a 10 minute gradient of 10 / 90% MeCN–water (each with
0.05% triuoroacetic acid, TFA) which showed two peaks at 4.6
and 4.8 minutes. These two compounds were puried by
preparative HPLC using a Phenomenex, Luna C18, 250 ꢂ 30 mm
column and a 40 minute gradient of 5 / 40% MeCN–water
(each with 0.05% TFA). The two diastereomers were isolated
separately, the HPLC fractions frozen at ꢁ80 ꢃC and lyophilized
to dryness. Combined yield ¼ 15.8 mg (96%). Diastereomer
eluting at 4.6 minutes: dH (500 MHz, CF3COOD) 1.49 (d, 6H, J ¼
Experimental
1. General
Chemicals were purchased from Sigma-Aldrich (Milwaukee, 5.9), 2.42 (m, 4H), 2.49 (s, 6H), 3.49 (br s, 4H), 4.05 (s, 3H), 5.02
Wisconsin, USA) unless indicated otherwise. The ionic liquid (br s, 2H), 7.85 (br s, 2H), 7.96 (d, 2H, J ¼ 9.6), 7.97, (s, 2H), 8.26
21860 | RSC Adv., 2014, 4, 21852–21863
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(d, 2H, J ¼ 9.4); MALDI-TOF MS m/z 690.6 (M + H)+; HRMS m/z
690.1675 (M + H)+ (690.1679 calculated). Diastereomer eluting
at 4.8 minutes: dH (500 MHz, CF3COOD) 1.50 (d, 6H, J ¼ 5.9),
2.42 (m, 4H), 2.50 (s, 6H), 3.49 (br s, 4H), 4.05 (s, 3H), 5.02 (br s,
2H), 7.85 (s, 2H), 7.95 (d, 2H, J ¼ 9.6), 7.96, (br d, 2H), 8.26 (d,
2H, J ¼ 9.5); MALDI-TOF MS m/z 690.5 (M + H)+; HRMS m/z
690.1671 (M + H)+ (690.1679 calculated).
Compound 6. 1,3-Dimethoxy-2-propanol was synthesized as
described in the literature.8 Crude 1,3-dimethoxy-2-propanol
(12.9 g, 0.108 mole) and potassium hydroxide (4.83 g, 0.08 mole)
ꢃ
was stirred at 80 C under a nitrogen atmosphere until all the
potassium hydroxide dissolved. Epichlorohydrin (1.85 g,
0.02 mole) was then added drop wise and the reaction was
heated at 80 ꢃC under a nitrogen atmosphere for 16 hours. The
reaction was then cooled to room temperature and quenched
with acetic acid (10 mL). The resulting suspension was poured
into ethyl acetate (150 mL) and aer thorough mixing it was
ltered to remove the precipitated solids. The ethyl acetate
extract was concentrated under reduced pressure to give a light
brown oil (8.06 g) which was puried by ash chromatography
using 70% hexanes, 25% ethyl acetate, 5% methanol. Product
fractions (Rf ¼ 0.2) were combined and concentrated under
reduced pressure to give a light yellow oil. Yield ¼ 2.46 g (42%).
nmax/cmꢁ1 3454 (OH), 1456, 1111; dH (500 MHz, CDCl3) 3.36
(s, 12H), 3.39–3.50 (m, 8H), 3.59 (d, 1H, J ¼ 6.5), 3.61 (d, 1H, J ¼
6.5), 3.64–3.71 (m, 4H), 3.74 (d, 1H, J ¼ 4.0), 3.93 (m, 1H);
MALDI-TOF MS m/z 319.2 (M + Na)+; HRMS m/z 319.1725 (M +
Na)+ (319.1733 calculated).
Compound 7. A solution of compound 1 (50 mg, 0.12 mmol),
compound 6 (140 mg, 0.48 mmol) and triphenylphosphine
(TPP, 126 mg, 0.48 mmol) was treated with triethylamine
(TEA, 0.098 mL, 0.72 mmol) and the solution was cooled in an
ice bath. Aer 10–15 minutes, diisopropylazodicarboxylate
(DIAD, 0.094 mL, 0.48 mmol) was added, the reaction was
stirred briey in the ice bath (10 minutes) and was then warmed
to room temperature and stirred under a nitrogen atmosphere.
Aer 3 hours, a small portion of the reaction was analyzed by
HPLC using a Phenomenex, C18 3.9 mm ꢂ 30 cm column and a
30 minute gradient of 10 / 100% MeCN–water (each with
0.05% triuoroacetic acid) at a ow rate of 1.0 mL per minute
and UV detection at 260 nm. Product was observed eluting at
25.2 minutes and was the major component. The reaction
mixture was then diluted with ethyl acetate (50 mL) and washed
with 1 N HCl (25 mL) followed by de-ionized water several times.
The ethyl acetate extract was then dried over anhydrous
magnesium sulfate and concentrated under reduced pressure.
The crude product was puried by ash chromatography on
silica using ethyl acetate as eluent. Yield ¼ 59.4 mg (51%).
nmax/cmꢁ1 1724 (CO), 1457, 1438,1230, 1118; dH (500 MHz,
CDCl3) 2.46 (s, 6H), 3.25 (s, 12H), 3.29 (s, 12H), 3.34–3.46 (m,
16H), 3.58–3.63 (m, 4H), 3.86–3.94 (m, 4H), 3.95 (s, 3H), 3.94–
3.98 (m, 4H), 4.73 (p, 2H, J ¼ 5.1), 7.54 (dd, 2H, J ¼ 9.4, 2.6), 7.72
(d, 2H, J ¼ 2.6), 7.90 (s, 2H), 8.15 (d, 2H, J ¼ 9.4); MALDI-TOF MS
m/z 974.8 (M + H)+; HRMS m/z 974.4758 (M + H)+ (974.4749
calculated).
Compound 4. A mixture of compound 1 (56 mg, 0.134
mmol), potassium carbonate (46 mg, 0.335 mmol), dibenzo-18-
crown-6 (121 mg, 0.335 mmol) and 1,3-butane sultone 2
(54.8 mg, 0.402 mmol) in 1-butyl-3-methylimidazolium hexa-
ꢃ
uorophosphate (2 mL) was heated at 120 C for 2 hours. The
reaction was cooled to room temperature and a small portion of
the reaction mixture was analyzed by HPLC as described above
which showed complete conversion to 3. To this reaction
mixture, 2,6-di-tert-butylpyridine (0.45 mL, 2.012 mmol) and
1,3-propane sultone (279 mg, 2.281 mmol) were added and the
ꢃ
reaction was heated at 155 C for 16 hours. The reaction was
cooled to room temperature and a small portion of the reaction
mixture was analyzed by HPLC which showed 62% conversion
to the N-sulfopropyl acridinium ester. The reaction mixture was
partitioned between 2 N HCl solution (20 mL) and ethyl acetate
(30 mL). The aqueous layer was separated and washed with
another portion of ethyl acetate (30 mL). The aqueous layer was
then reuxed for 4 hours and analyzed by HPLC which showed
complete hydrolysis to 4. The crude product was puried by
preparative HPLC using a Phenomenex, Luna C18, 250 ꢂ 30 mm
column and a 40 minute gradient of 5 / 40% MeCN–water
(each with 0.05% TFA). The product 4 (containing two diaste-
reomers) fractions were collected and frozen at ꢁ80 ꢃC and
lyophilized to dryness. Yield ¼ 39.4 mg (37%). nmax/cmꢁ1 3364
(OH), 1748 and 1712 (CO), 1613, 1468, 1223, 1149; dH (500 MHz,
CF3COOD) 1.60 (d, 6H, J ¼ 5.9), 2.53 (m, 4H), 2.63 (s, 6H), 2.97
(t, 2H, J ¼ 6.3), 3.62 (m, 4H), 3.87 (t, 2H, J ¼ 6.2), 5.11 (t, 2H, J ¼
6.2), 5.86 (t, 2H, J ¼ 8.9), 7.95 (d, 2H, J ¼ 2.7), 8.14 (s, 2H), 8.19
(dd, 2H, J ¼ 9.9, 2.6), 8.87 (d, 2H, J ¼ 10.0); MALDI-TOF MS m/z
798.2 (M + H)+; HRMS m/z 798.1539 (M + H)+ (798.1560
calculated).
Compound 5. A solution of compound 4 (23 mg, 0.028
mmol) in DMF (2 mL) was treated with diisopropylethylamine
(0.025 mL, 0.142 mmol) and N,N,N0,N0-tetramethyl-O-(N-succi-
nimidyl)uronium tetrauoroborate (TSTU,13 mg, 0.042 mmol).
The reaction was stirred at room temperature for 1 hour. HPLC
analysis of the reaction mixture showed complete conversion to
its NHS ester 5. The product was puried by preparative HPLC
using a Phenomenex, Luna C18, 250 ꢂ 30 mm column and a 40
minute gradient of 10 / 40% MeCN–water (each with 0.05%
TFA). The product fractions conꢃtaining 5 (containing two dia-
stereomers) were frozen at ꢁ80 C and lyophilized to dryness.
Yield ¼ 9.4 mg (37%). nmax/cmꢁ1 1742 and 1686 (CO), 1469,
1208, 1137; dH (500 MHz, CF3COOD) 1.50 (d, 6H, J ¼ 6.0), 2.43
(m, 4H), 2.53 (s, 6H), 2.87 (m, 2H), 3.12 (s, 4H), 3.54 (m, 4H),
3.77 (t, 2H, J ¼ 6.7), 4.98 (t, 2H, J ¼ 6.5), 5.77 (m, 2H), 7.84 (d, 2H,
J ¼ 2.7), 8.05 (s, 2H), 8.09 (dd, 2H, J ¼ 9.9, 2.6), 8.77 (d, 2H, J ¼
10.0); MALDI-TOF MS m/z 895.1 (M + H)+; HRMS m/z 895.1735
(M + H)+ (895.1724 calculated).
Compound 8. A mixture of compound 7 (59 mg, 0.061
mmol), distilled 1,3-propane sultone (0.110 g, 0.91 mmol) and
2,6-di-tert-butylpyridine (0.067 mL, 0.31 mmol) in 1-butyl-3-
methylimidazolium hexauorophosphate (1 mL) was heated at
ꢃ
150 C for 24 hours. The reaction was cooled at room temper-
ature and a small portion of the reaction mixture was analyzed
by HPLC as described above. Approximately 65% conversion to
the N-sulfopropyl acridinium ester was noted eluting at 20
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minutes in a clean reaction. The reaction was diluted with ethyl by preparative HPLC as described earlier. The HPLC fractions
acetate (5 mL) and applied to a silica column equilibrated with containing 10 were frozen at ꢁ80 ꢃC and lyophilized to dryness.
ethyl acetate. The column was rst eluted with ethyl acetate Yield ¼ 12.3 mg (67%). nmax/cmꢁ1 1815, 1781, 1741 and 1689
(500 mL) to elute unreacted starting material and base followed (CO), 1613, 1468, 1204, 1136; dH (500 MHz, CD3COOD) 2.53
by 1 : 1 ethyl acetate–methanol (500 mL) to elute product. The (t, 2H, J ¼ 7.4), 2.71 (s, 6H), 2.80 (t, 2H, J ¼ 7.2), 2.84–2.91 (m,
product fractions were combined and concentrated under 2H), 2.98 (s, 4H), 3.37 (s, 12H), 3.42 (s, 12H), 3.53–3.64 (m, 18H),
reduced pressure. The N-sulfopropyl acridinium methyl ester 3.76 (t, 2H, J ¼ 5.4), 3.79–3.93 (m, 14H), 4.05–4.17 (m, 8H), 5.07
was reuxed in 1 N HCl (10 mL) for 2 hours. The reaction was (m, 2H), 5.95 (m, 2H), 7.97 (s, 2H), 8.04 (d, 2H, J ¼ 2.6), 8.36 (dd,
then cooled to room temperature and analyzed by HPLC which 2H, J ¼ 10.0, 2.7), 9.08 (d, 2H, J ¼ 10.1); MALDI-TOF MS m/z
showed clean formation of product 8 eluting at 17.8 minutes. 1423.6 (M + H)+; HRMS m/z 1423.6245 (M + H)+ (1423.6217
The product was puried by preparative HPLC using an YMC, calculated).
C
18, 30 ꢂ 250 mm column and the same gradient used for
analytical analysis. Product fractions containing were
8
3. Synthesis of BSA conjugates of acridinium esters (Table 1)
combined and concentrated under reduced pressure. Yield ¼ 34
mg (51%). nmax/cmꢁ1 1752 and 1714 (CO), 1613, 1468, 1148; dH
(500 MHz, CF3COOD) 2.54 (s, 6H), 2.88 (m, 2H), 3.46 (s, 12H),
3.50 (s, 12H), 3.72 (m, 16H), 3.79 (m, 2H), 3.93 (m, 4H), 4.06 (m,
8H), 4.98 (p, 2H, J ¼ 4.8), 5.78 (m, 2H), 7.98 (d, 2H, J ¼ 2.6), 8.06
(s, 2H), 8.16 (dd, 2H, J ¼ 10.1, 2.6), 8.81 (d, 2H, J ¼ 10.0); MALDI-
TOF MS m/z 1082.4 (M + H)+; HRMS m/z 1082.4674 (M + H)+
(1082.4631 calculated).
BSA (1 mg, 15 nanomoles, 0.2 mL of a 5 mg mLꢁ1 solution) was
diluted with 0.2 mL of 0.1 M sodium carbonate, pH 9. The
protein solutions, in separate labeling reactions, were treated
with ve equivalents of various acridinium esters of Fig. 3. The
acridinium esters were dissolved in dimethyl sulfoxide to give
2 mg mLꢁ1 solutions. For labeling with the various acridinium
esters, 5 equivalents of the DMSO solution was added. The
reactions were stirred at room temperature for 2 hours. The
reactions were then diluted with 0.5 mL de-ionized water and
transferred to 4 mL Amicon lters from Millipore (MW 30 000
cutoff). The labeling reactions in the lters were further diluted
with 3 mL de-ionized water. The solutions were concentrated to
ꢀ0.1 mL be centrifugation at 7000g for 10 minutes. This dilu-
tion and concentration process was repeated three more times.
The nal conjugate solutions were transferred to vials and the
solutions were frozen and lyophilized. The lyophilized conju-
gates were dissolved in 0.5 mL of de-ionized water. The conju-
gates were analyzed by MALDI-TOF mass spectrometry to
measure acridinium ester incorporation. This entailed
measuring the molecular weight of the unlabeled protein and
the labeled protein. The acridinium ester label contributed to
the observed difference in mass between these two measure-
ments. By knowing the molecular weight of the specic acridi-
nium ester label, the extent of label incorporation for that
specic acridinium ester could thus be calculated (Table 1).
Compound 9. A solution of compound 8 (34 mg, 0.031
mmol) in DMF (2 mL) was treated with diisopropylethylamine
(0.0082 mL, 0.047 mmol) and N,N,N0,N0-tetramethyl-O-(N-suc-
cinimidyl)uronium tetrauoroborate (TSTU, 14.2 mg, 0.047
mmol). The reaction was stirred at room temperature. Aer 30
minutes HPLC analysis as described above indicated complete
conversion to the N-hydroxysuccinimide ester eluting at 19.3
min. This solution was added drop wise to a stirred solution of
2,20-(ethylenedioxy)bis(ethylamine) (0.047 mL, 10 equivalents)
dissolved in DMF (2 mL). The resulting solution was stirred at
room temperature. Aer 30 minutes, HPLC analysis indicated
complete conversion to product 9 eluting at 14.6 minutes. The
product was puried by preparative HPLC as described above.
HPLC fractions were concentrated under reduced pressure.
Yield ¼ 28.4 mg (74%). nmax/cmꢁ1 1751 and 1689 (CO), 1613,
1468, 1223, 1163, 1117; dH (500 MHz, CD3COOD) 2.71 (s, 6H),
2.88 (m, 2H), 3.37 (s, 12H), 3.43 (s, 12H), 3.53–3.64 (m, 18H),
3.80–3.95 (m, 14H), 4.05–4.17 (m, 8H), 5.07 (m, 2H), 5.95 (m,
2H), 7.97 (s, 2H), 8.04 (d, 2H, J ¼ 2.7), 8.36 (dd, 2H, J ¼ 10.0, 2.7),
9.08 (d, 2H, J ¼ 10.1); MALDI-TOF MS m/z 1212.9 (M + H)+;
HRMS m/z 1212.5729 (M + H)+ (1212.5737 calculated).
4. Chemiluminescence measurements (Table 1, Fig. 6 and 7)
Chemiluminescence of BSA conjugates of acridinium esters
Compound 10. A solution of compound 9 (15.4 mg, 0.013 (Fig. 3) was measured on an AutoLumat LB953 Plus lumin-
mmol) in methanol (2 mL) was treated with diisopropylethyl- ometer from Berthold Technologies. The conjugates, 2 mg
amine (0.011 mL, 5 equivalents) and glutaric anhydride (0.0072 mLꢁ1, were serially diluted 105-fold for chemiluminescence
mg, 5 equivalents). The reaction was stirred at room tempera- measurements in an aqueous buffer of 10 mM disodium
ture. Aer 30 minutes, HPLC analysis as described above hydrogen phosphate, 0.15 M NaCl, 8 mM sodium azide and
showed complete conversion to the glutarate derivative eluting 0.015 mM bovine serum albumin (BSA), pH ¼ 8.0. Samples of
at 16.2 minutes. The reaction mixture was concentrated under 0.010 mL volumes of each diluted acridinium ester were
reduced pressure. The residue was suspended in anhydrous dispensed into the bottom of cuvettes. Cuvettes were placed
toluene (5 mL) and was evaporated to dryness. The crude glu- into the primed LB953 and the chemiluminescence reaction
tarate derivative was dissolved in DMF (2 mL) and treated with was initiated with the sequential addition of 0.3 mL of reagent
diisopropylethylamine (0.022 mL, 10 equivalents) and TSTU 1, a solution of 0.5% hydrogen peroxide in 0.1 M nitric acid
(38 mg, 10 equivalents). The reaction was stirred at room followed by an almost instantaneous (<1 second delay) addition
temperature. Aer 15 minutes, HPLC analysis of the reaction of 0.3 mL of reagent 2, a solution of 0.25 M sodium hydroxide
mixture showed the NHS ester 10 as the major product (ꢀ80% with or without surfactant. For measurements with CTAC, 7 mM
conversion) eluting at 17.2 minutes. The product was puried of this cationic surfactant was included in reagent 2. In all
21862 | RSC Adv., 2014, 4, 21852–21863
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measurements, the nal pH of the chemiluminescence reaction
was basic (0.15 M NaOH).
References
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intervals of 0.1 second (20 seconds total time), in the presence
of CTAC or 240 intervals of 0.5 seconds (2 minutes total time), in
the absence of CTAC from the point of chemiluminescence
initiation with the addition of 0.25 M NaOH. Each chem-
iluminescence reaction was carried out a minimum of three
times, averaged and converted to a percentage of the chem-
iluminescence accumulated up to each time interval. The
output from the luminometer instrument was expressed as RLU
(Relative Light Unit).
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The acridinium ester labeled BSA conjugates were diluted to a
concentration of 0.2 nM in a buffer of in an aqueous buffer of 10
mM phosphate, 0.15 M NaCl, 8 mM sodium azide and 0.015
mM bovine serum albumin (BSA), pH ¼ 6.0 or 7.4. Two milliliter
volumes of the diluted conjugates were sealed in glass vials and
˙
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ꢃ
incubated at 25 C for ve weeks protected from light. Chem-
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iluminescence of 0.01 mL samples (ve replicates) of the diluted
conjugates was periodically measured as a function of time.
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AutoLumat Plus LB953 luminometer. The averaged results were
calculated as residual chemiluminescence percentages with
respect to values determined at day 1.
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´
´
K. Krzyminski, A. O˙zog, P. Malecha, A. D. Roshal,
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UV-visible spectrophotometry of acridinium esters 5, 10, 11 and
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transition was carried out by dissolving the HPLC-puried
acridinium esters in DMSO (ꢀ1 mg mLꢁ1). These solutions were
further diluted 20-fold into 0.2 M phosphate or borate buffer at
pH 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0 (pH 10 was 0.2 M
borate buffer). The diluted acridinium ester solutions were
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absorption intensity of the long wavelength acridinium
absorption band at ꢀ410 nm was measured as a function of pH.
A plot of this data is shown in Fig. 8 and 9.
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