A comparison of chemiluminescent acridinium dimethylphenyl ester labels with different conjugation sites

Article abstract of DOI:10.1039/c4ob02528h

Chemiluminescent acridinium dimethylphenyl esters are highly sensitive labels that are used in automated assays for clinical diagnosis. Light emission from these labels and their conjugates is triggered by treatment with alkaline peroxide. Conjugation of

Full text of DOI:10.1039/c4ob02528h

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A comparison of chemiluminescent acridinium
dimethylphenyl ester labels with different
conjugation sites†
Cite this: DOI: 10.1039/c4ob02528h
Anand Natrajan* and David Wen
Chemiluminescent acridinium dimethylphenyl esters are highly sensitive labels that are used in automated
assays for clinical diagnosis. Light emission from these labels and their conjugates is triggered by treat-
ment with alkaline peroxide. Conjugation of acridinium ester labels is normally done at the phenol. During
the chemiluminescent reaction of these acridinium esters, the phenolic ester is cleaved and the light
emitting acridone moiety is liberated from its conjugate partner. In the current study, we report the syn-
thesis of three new acridinium esters with conjugation sites at the acridinium nitrogen and compare their
properties with that of a conventional acridinium ester with a conjugation site at the phenol. Our study is
the first that provides a direct comparison of the emissive properties of acridinium dimethylphenyl esters
(free labels and protein conjugates) with different conjugation sites, one where the light emitting acridone
remains attached to its conjugate partner versus conventional labeling which results in cleavage of the
acridone from the conjugate. Our results indicate that the conjugation at the acridinium nitrogen, which
also alters how the acridinium ring and phenol are oriented with respect to the protein surface, has a
minimal impact on emission kinetics and emission spectra. However, this mode of conjugation to three
different proteins led to a significant increase in light yield which should be useful for improving the assay
sensitivity.
Received 2nd December 2014,
Accepted 2nd January 2015
DOI: 10.1039/c4ob02528h
www.rsc.org/obc
nitric acid containing 0.5% hydrogen peroxide followed by the
addition of 0.25 M sodium hydroxide containing the cationic
Introduction
Chemiluminescent acridinium dimethylphenyl esters1 are surfactant cetyltrimethylammonium chloride (CTAC).1e The
highly sensitive labels that are used in automated immuno- surfactant accelerates light emission from the labels by provid-
assays for clinical diagnosis. Conjugates of these labels are used ing a high local concentration of reactive hydroperoxide ions
along with magnetic microparticles for the measurement of a at the cationic micellar surface.1e Micelles of CTAC also
wide range of clinically important analytes.1a–d Light emission provide a low-polarity environment1e,3 which is conducive to
from acridinium ester labels and their conjugates is typically the formation of dioxetanes and/or dioxetanones that have
triggered with alkaline peroxide. Acridinium esters at physio- been proposed as reaction intermediates in the chemilumines-
logical pH exist as non-chemiluminescent water adducts (com- cence pathway of acridinium esters.4 Ultimately, excited state
monly referred to as pseudobases1g,h,2) that are formed by the acridone is believed to be the light emitting moiety respon-
addition of water at C-9 of the acridinium ring (see Fig. 1 for sible for the acridinium ester chemiluminescence.4
the numbering system). Consequently, an acid pre-treatment is
Conjugation of acridinium esters to biomolecules such as
required to convert the pseudobases to the acridinium forms proteins can potentially be carried out at different sites of the
before the chemiluminescence can be initiated from acridi- acridinium ester molecule as illustrated in Fig. 1, panel A. For
nium ester labels and their conjugates. In practice, at the end immunoassay applications, conjugation of acridinium
of each immunoassay on our automated instruments, chemi- dimethylphenyl ester labels is typically carried out at the
luminescence is triggered by the sequential addition of 0.1 M phenol by placement of a reactive group as illustrated in struc-
ture 4, which has an N-hydroxysuccinimide active ester
appended to a polar, zwitterion-containing linker (Fig. 1, panel
Siemens Healthcare Diagnostics, Advanced Technology and Pre-Development, 333
B).1g In fact, conjugation at the phenol is the most commonly
Coney Street, East Walpole, MA 02032, USA. E-mail: anand.natrajan@siemens.com;
described strategy in the literature for applications in immu-
Fax: +1-508-660-4591; Tel: +1-508-660-4582
†Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ob02528h
noassays as well as probe assays.1,5 This is because of two
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Fig. 1 Panel A: general structure of a chemiluminescent acridinium dimethylphenyl ester illustrating pseudobase-acridinium equilibrium. Potential
sites for conjugation are indicated by the arrows. In the current study, conjugation sites were incorporated at R1 at the acridinium nitrogen using sul-
fonylpentafluorophenyl chemistry.1h Panel B: structure of a high light yield, N-sulfopropyl acridinium dimethylphenyl ester with a C-2 isopropoxy
group and a hydrophilic, zwitterionic linker containing a conjugation site attached to the phenol.1g
main reasons. First, incorporating a conjugation site at the
Conjugation of acridinium esters to various molecules,
phenol is relatively easy from a synthesis point of view. either at the acridinium ring carbons or at the acridinium
In addition, this approach leaves the acridinium ring intact for nitrogen (Fig. 1, panel A), is synthetically more challenging but
suitable chemical modification to both alter the emissive pro- has also been described in the literature.6 For example, syn-
perties of the label and influence its partitioning into CTAC theses of conjugates of acridinium dimethylphenyl esters to
micelles. For example in structure 4 (Fig. 1, panel B), the iso- various fluorescent dyes linked either through the acridinium
propoxy group at C-2 of the acridinium ring not only induces a ring at C-2/C-3 or the acridinium nitrogen have been descri-
bathochromic shift in the emission wavelength but also bed.6a These conjugates showed very efficient energy transfer
increases the light yield twofold compared to the corres- from excited state acridone to the attached dye when their
ponding unsubstituted label.1g Moreover, the isopropoxy chemiluminescence is triggered with alkaline peroxide. A range
group increases the hydrophobicity of the acridinium ring of chemiluminescent, acridinium ester conjugates of fluorescent
which results in its strong partitioning into CTAC micelles.1g dyes emitting at different wavelengths was described using
The other reason why acridinium ester conjugation is typically this strategy.6a Acridinium dimethylphenyl ester labels with
carried out at the phenol is because during light emission, the conjugation sites at C-2 have also been shown to be potentially
phenolic ester is cleaved and the light emitting acridone useful for homogeneous immunoassays using resonance
moiety is free to diffuse away from its conjugate partner such energy transfer.6b
as a protein as illustrated in Fig. 2, panel B. Thus, the impact
Synthetic access to acridinium esters with conjugation sites
of the protein on chemiluminescence is expected to be at C-2 or C-3, although somewhat tedious, is relatively straight-
minimal. On the other hand if conjugation is carried out forward but functionalization of the acridine nitrogen with
either at the acridinium nitrogen (illustrated in Fig. 2, panel A) complex alkylating reagents is much more difficult due to
or at other sites on the acridinium ring, then following the steric hindrance. Conjugation through the acridinium nitro-
chemiluminescence reaction, the light emitting acridone will gen, nevertheless, is attractive because it leaves C-2 and C-3 in
remain attached to the protein and its chemiluminescence can the acridinium ring free for modification with other functional
be potentially affected.
groups such as in 4 (Fig. 1). Functionalization of the acridine
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Fig. 2 Fate of the light-emitting, acridone moiety following chemiluminescent reaction of a protein conjugate of an acridinium dimethylphenyl
ester. Panel A: when conjugation is performed at the acridinium ring (illustrated for the acridinium nitrogen) the light emitting acridone species
remains attached to the protein. Panel B: when conjugation is performed at the phenol, excited state acridone is cleaved from the protein and can
diffuse away from the protein surface.
nitrogen in variable yields has been described in the literature assemble a hydrophilic, charge-neutral acridinium ester which
using reagents such as tert-butyl iodoacetate or other simple exhibited high light yield and low non-specific binding to mag-
alkyl iodides and bromides as both the alkylating reagent and netic microparticles.1h In the current study, we report the syn-
solvent.7a,b N-Alkylation of the acridine nitrogen in acridine thesis of three new acridinium esters with conjugation sites at
esters and related compounds with powerful trifluoromethane- the acridinium nitrogen using N-sulfonyl pentafluorophenyl
sulfonates (triflates) as alkylating reagents has been reported ester chemistry and our simple synthetic protocol should prove
to afford better yields in a few instances8 although in more useful for the assembly of other acridinium esters with
recent studies,9 reported yields were quite poor for these complex N-alkyl functional groups. We provide a comparison
N-alkylation reactions even with very reactive alkylating reagents. of these new acridinium esters and their protein conjugates to
Consistent with these later studies,9 in our experience, with three different proteins with different isoelectric points (pI),
the exception of strong methylating reagents or 1,3-propane with a structurally analogous acridinium ester 4 which has a
sultone,1 the N-alkylation of acridine dimethylphenyl ester pre- conjugation site at the phenol. Our study is the first that pro-
cursors is very difficult to achieve in good yields even with vides a direct comparison of the properties of acridinium
powerful alkylating reagents such as trifluoromethanesulfo- dimethylphenyl esters (free labels and protein conjugates)
nates. On the other hand, introduction of the N-sulfopropyl with different conjugation sites. Conjugation at the acridinium
group can be conveniently carried out in a wide range of acri- nitrogen reverses the orientation of the acridinium ring and
dine dimethylphenyl ester substrates in excellent yields by phenol with respect to the protein surface compared to label-
using room temperature ionic liquids as reaction media and ing at the phenol (Fig. 2). In addition, in the former, the light
using commercially available 1,3-propane sultone as the alky- emitting acridone moiety remains attached to its conjugate
lating reagent.1f–j,10
partner versus conventional labeling which results in cleavage
We reported recently that the N-sulfopropyl group in a 2-iso- of the acridone from the conjugate. Our results provide insight
propoxy-substituted acridinium ester can easily be converted into the contributions of the microenvironment of the cationic
to the activated but stable sulfonyl pentafluorophenyl ester via surfactant CTAC and proteins in affecting the acridinium ester
the sulfonyl chloride.1h We used this pentafluorophenyl ester to chemiluminescence.
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the sulfonate moiety in the N-sulfopropyl acridinium ester to
the acid chloride using thionyl chloride followed by conden-
sation with pentafluorophenol. Compared to the sulfonyl chloride,
Results and discussion
Synthesis of acridinium esters and conjugates
The synthesis of three acridinium dimethylphenyl esters 3a–3c the sulfonyl pentafluorophenyl ester 1 is much more stable. It
containing a C-2 isopropoxy group and with three different can conveniently be purified by preparative HPLC (if required)
linkers attached to the acridinium nitrogen is illustrated in and is stable to storage under anhydrous conditions at low tem-
Fig. 3. The syntheses were accomplished using the common peratures (∼−10 °C) for an extended period of time (months).
intermediate 1 whose synthesis we have described recently.1h
We wanted to determine whether the length and nature of
The synthesis of 1 is straightforward and entails conversion of the linker attached to the acridinium nitrogen would affect its
Fig. 3 Synthetic scheme for compounds 3a–3c with conjugation sites at the acridinium nitrogen. (a) 1 : 1, DMF–MeCN, 75 °C; (b) DMSO, diisopropyl-
ethylamine (DIPEA) 75 °C; (c) disuccinimidyl glutarate (DSG), 0.1 M sodium phosphate pH = 7.2/MeCN.
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corresponding labels 3a–3c and 4). Similarly, protein conju-
gates of the labels 3a–3c and 4 were used for chemilumines-
cence measurements. Both the free labels as well as the
protein conjugates were also used for recording emission
spectra. The output of the luminometer was in RLUs (Relative
Light Units) whereas emission spectra recorded using the
spectral camera had an output of emission intensity (lumi-
nance) as a function of emission wavelength.
Table 1 Label incorporation in proteins using 10 equivalents input of
acridinium ester labels 3a–3c and 4 in the labeling reactions. Label
incorporation (rounded to the nearest integer) was measured by mass
spectroscopy
Label
BSA
Anti-TSH Mab
Anti-HBsAg Mab
4
6.0
5.0
4.0
7.0
6.0
4.0
4.0
8.0
8.0
3.0
6.0
6.0
3a
3b
3c
For chemiluminescence measurements of the free labels,
the amine derivatives 2a–2c (precursors to the NHS esters 3a–
3c) and the amine precursor of 4 were dissolved in dimethyl
sulfoxide (0.25–0.5 mM) and these solutions were serially
diluted to 0.25–0.5 nM concentrations in buffer. Chemilumi-
nescence from 0.01 mL samples was initiated by the addition
of 0.3 mL of 0.1 M nitric acid containing 0.5% hydrogen per-
oxide followed by the instantaneous addition of 0.25 M
sodium hydroxide with or without CTAC (7 mM). In the pres-
ence of CTAC, emission was fast (similar to what we have
reported previously1e) and light was collected for a period of 10
seconds integrated at 0.1 second intervals (five replicates). In
the absence of CTAC, light emission from acridinium
dimethylphenyl esters is typically slow1e and therefore light
was collected for a period of 2 minutes integrated at 0.5
second intervals. Chemiluminescence measurements of
protein conjugates were carried out in a similar manner.
Protein conjugates (∼2 mg mL−1) were serially diluted to
0.1–0.2 nM and the chemiluminescence from 0.01 mL samples
was measured as described above. Additional details can be
found in the Experimental section.
chemiluminescence. In addition, we wanted to maintain ade-
quate aqueous solubility of the acridinium esters. Towards
that end, three different linkers, two i and ii derived from poly
(ethylene)glycol and one iii containing a sulfobetaine zwitter-
ion were selected for reaction with 1. Linker i is commercially
available and we have reported the syntheses of ii and iii in
our earlier work.1b,e Displacement of the pentafluorophenyl
ester in 1 by i–iii was easily accomplished by heating 1 with a
5-fold excess of the three linkers to give the amine derivatives
2a–2c. These displacement reactions proceeded cleanly with
minimal hydrolysis of the pentafluorophenyl ester 1. Following
HPLC purification of 2a–2c, they were converted to the
N-hydroxysuccinimide esters 3a–3c using standard chemistry
followed by HPLC purification. Yields of all compounds following
HPLC purification were generally quite good as detailed in
the Experimental section and in Fig. 3. HPLC traces and
NMR spectra of compounds 2a–2c and 3a–3c are shown in
Fig. S1–S6 (ESI†).
Protein conjugates were prepared using labels 3a–3c and 4,
the latter representing a conventional acridinium ester with
the same C-2 isopropoxy group but with the conjugation site at
the phenol as described earlier. Three different proteins were
selected, BSA (BSA = bovine serum albumin, a small acidic
protein), an anti-TSH monoclonal antibody1e (Mab) with an
acidic pI of 5.6 (TSH = thyroid stimulating hormone) and an
anti-HBsAg Mab1e with a neutral pI of 6.9 (HBsAg = hepatitis B
surface antigen). Label incorporation in these three different
proteins (Table 1) was measured by mass spectroscopy. Some
differences were noted in the labeling efficiency between the
four labels with 3a and 3b leading to slightly lower levels of
incorporation compared to 4 and 3c which have more polar,
zwitterion-containing linkers. For a given acridinium ester
however, label incorporation across the three different proteins
was quite similar. For example 3c gave 7, 8 and 6 incorporated
labels respectively in BSA, anti-TSH Mab and anti-HBsAg Mab.
Thus, protein pI did not play a significant role in labeling
efficacy for a particular label.
Chemiluminescence emission profiles in the presence of
CTAC of the free labels 2a–2c, the amine precursor of 4 as well
as the three protein conjugates of the four labels 3a–3c and 4
are shown in Fig. 4. The corresponding emission profiles in
the absence of CTAC are also shown in Fig. S7–S10 (ESI†). As
can be noted from Fig. 4, both the free labels as well as the
protein conjugates of the various labels exhibited fast light
emission in the presence of CTAC with total emission times of
<5 seconds regardless of the label or the protein. This fast
emission is typical of acridinium dimethylphenyl esters1e and
the emission profiles illustrated in Fig. 4 indicate that the con-
jugation site does not play any significant role in emission
kinetics. The fast light emission also implies that the acridi-
nium rings in the various labels as well as in their conjugates
are able to partition equally well into CTAC micelles which are
responsible for accelerating light emission.1e In the absence of
CTAC, light emission from the free labels as well as the protein
conjugates was slow, taking up to a minute for complete emis-
sion as illustrated in Fig. S7–S10 (ESI†).
Emission spectra of the labels and protein conjugates are
shown in Fig. 5 and S11 and S12 (ESI†). For compound 4, the
Chemiluminescence measurements
Chemiluminescence measurements of the free labels, amine conjugation site is at the phenol and the acridone is released
derivatives 2a–2c as well as the amine precursor1g of 4 were during the chemiluminescence reaction following cleavage of
carried out using a luminometer and a spectral camera as the phenolic ester bond. Consequently, any change in the
described in the Experimental section. (The amine derivatives emission spectrum of the acridone is unlikely for the conju-
2a–2c were used to avoid hydrolysis of the N-hydroxysuccini- gate and Fig. 5 (panel A) confirms this expectation. Both the
mide esters that are appended to the zwitterionic linker in the free label and the BSA conjugate showed emission from the
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Fig. 4 Chemiluminescence emission profiles. Panel A: free labels, the amine precursor of 4 and amine precursors 2a–2c. Panel B: BSA conjugates
of compound 4 and 3a–3c. Panel C: anti-TSH Mab conjugates of compound 4 and 3a–3c. Panel D: anti-HBsAg Mab conjugates of compound 4 and
3a–3c. Chemiluminescence was initiated by the sequential addition of 0.3 mL of 0.1 M nitric acid containing 0.5% hydrogen peroxide followed by
0.3 mL of 0.25 M sodium hydroxide containing 7 mM CTAC (CTAC = cetyltrimethylammonium chloride). Fast light emission was observed regardless
of the conjugation site for both free labels and protein conjugates.
2-isopropoxy-substituted acridone1h with an emission maximum (with CTAC). For label 4 with a conjugation site at the phenol,
centered at 452–454 nm. For the free labels 2a–2c, the same the specific activity was observed to be relatively unchanged
2-isopropoxy-substituted acridone is formed in the reaction from the free label to its protein conjugates. Thus the specific
although with different N-alkyl groups. All three labels 2a–2c activity of the free label was observed to be 5.4 (units of 1019
showed identical emission spectra compared to the amine pre- RLUs per mole) whereas the corresponding values were 5.3, 5.1
cursor of 4 with emission maxima centered at 454 nm. These and 4.4 for BSA, anti-TSH Mab and anti-HBsAg Mab conju-
results are consistent with our earlier study where we noted gates, respectively. In comparison, the free labels 2a–2c
that the structure of the N-alkyl group in a 2-isopropoxy-substi- showed a slight drop in specific activity when compared to 4
tuted, charge-neutral acridinium ester had no impact on its as shown in Fig. 6. However unlike 4, conjugation of the NHS
emission spectrum.1h The BSA conjugates of the labels 3a–3c esters 3a–3c (corresponding to the amine derivatives 2a–2c) to
interestingly also showed very similar emission spectra to the the three proteins showed a consistent increase in light yield
free labels (Fig. 5, panels B–D). These observations indicate compared to the free labels as can be noted from Fig. 6. These
that even when the light emitting acridone moieties from observations may appear counterintuitive because the excited
these labels are constrained on the protein, their emission state acridone moieties from 3a–3c are constrained on the pro-
spectra are largely unaffected. Similarly, the antibody conju- teins and could potentially be subjected to quenching by the
gates of 4 and 3a–3c showed identical emission spectra regard- latter’s aromatic amino acids. However, all three labels emit
less of the conjugation site as illustrated in Fig. S11 and S12 light with wavelength maxima at 454 nm which is well separ-
(ESI†).
ated from the dominant absorption band of BSA and anti-
Is the light yield affected when conjugation of acridinium bodies at 280 nm. Consequently quenching by energy transfer
dimethylphenyl esters is performed using functional groups at is expected to be unlikely which is consistent with our
the acridinium nitrogen rather than at the phenol? To answer observations.
this question, we measured the specific chemiluminescence
To better understand the mechanism of the enhancement
activity of the free labels as well their protein conjugates (both in light yield for the protein conjugates of 3a–3c, we calculated
with and without CTAC) and the results are shown in Fig. 6 the relative change in light yield caused by protein conjugation
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Fig. 5 Emission spectra. Panel A: 4 (amine precursor) and the BSA conjugate of 4. Panel B: 2a and the BSA conjugate of 3a. Panel C: 2b and the BSA
conjugate of 3b. Panel D: 2c and the BSA conjugate of 3c. Emission spectra were obtained on an FSSS (Fast Spectral Scanning System) using a
PR-740 spectroradiometer (camera) from Photo Research Inc. Emission spectra of acridinium esters and BSA conjugates did not change regardless
of the conjugation site and even when excited state acridones from 3a–3c were constrained on the protein.
Fig. 6 Panel A: observed specific chemiluminescence activity (SCA) in units of 1019 RLUs per mole of acridinium ester labels and their protein conju-
gates (average of five replicates). Panel B: relative change in the light yield caused by protein conjugation. Specific activities of the free labels (amine
precursor of 4 and 2a–2c) were assigned a value of one. Conjugation of the labels 3a–3c (NHS esters corresponding to 2a–2c) through conjugation
sites at the acridinium nitrogen led to an increase in the light yield in the protein conjugates. A corresponding increase was not observed when con-
jugation was performed at the phenol for 4.
(free labels versus conjugates) as illustrated in Fig. 6, panel without CTAC. Table 2 also lists the ratios of the enhance-
B. In panel B, the specific activity of each free label (2a–2c and ments in light yield caused by CTAC for conjugates versus free
amine derivative of 4) was assigned a value of one. In addition, labels.
in Table 2, we have listed the observed enhancements in light
An examination of Fig. 6 and Table 2 reveals some interest-
yields for the free labels and the protein conjugates in the ing differences between the different labels and their protein
presence of CTAC compared to the control experiments conjugates while at the same time illustrating the complexity
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different surfactant-mediated enhancements in conjugates
versus free labels. Similar arguments apply for 3b and 3c.
Thus, an increased light yield from the protein conjugates of
3a–3c appears to be due to a combination of two factors: (a)
intrinsically higher light yield in the conjugates in the
absence of CTAC especially for the BSA conjugates, and (b)
greater CTAC enhancement in the light yield for the antibody
conjugates.
Table 2 Observed enhancement (fold) in the chemiluminescence of
free labels and protein conjugates in the presence of CTAC. Light emis-
sion was triggered by the sequential addition of 0.3 mL of 0.1 M nitric
acid containing 0.5% hydrogen peroxide followed by 0.25 M sodium
hydroxide with or without CTAC (control experiment). The light yield in
the absence of the surfactant was assigned a value of one for each label
and conjugate. Values in parentheses are the ratios in CTAC enhance-
ments of conjugates versus free labels (assigned a relative value of one)
CTAC enhancement,
In our earlier work,1e we proposed that surfactant-mediated
enhancements in the light yield of acridinium dimethylphenyl
esters and their conjugates result from the less polar micellar
environment which is conducive to the formation of the
immediate precursors (dioxetane and/or dioxetanone) of
excited state acridone. Obviously, this requires strong parti-
tioning of the acridinium ring into surfactant micelles and we
provided evidence that this partitioning results from a combi-
nation of hydrophobic and charge interactions.1g Our current
results indicate that acridinium dimethylphenyl esters with
conjugation sites at the acridinium nitrogen and their protein
conjugates are also equally capable of partitioning into surfac-
tant aggregates. In addition to the micellar environment, the
environment of the protein also appears to play a role in
affecting the light yield from these labels compared to conven-
tional acridinium ester labels with conjugation sites at the
Free label/conjugate
(conjugate/free label)
4, amine precursor1g
2a
2b
2c
BSA-4
BSA-3a
BSA-3b
BSA-3c
anti-TSH Mab-4
anti-TSH Mab-3a
anti-TSH Mab-3b
anti-TSH-Mab-3c
anti-HBsAg Mab-4
anti-HBsAg Mab-3a
anti-HBsAg Mab-3b
anti-HBsAg Mab-3c
4.1
4.3
3.7
4.3
3.7 (0.9)
4.8 (1.1)
4.5 (1.2)
4.0 (0.9)
4.6 (1.1)
5.9 (1.4)
5.4 (1.5)
6.2 (1.4)
6.1 (1.5)
6.1 (1.4)
5.0 (1.4)
5.9 (1.4)
of the effects leading to these observed differences. For label 4, phenol. The combined environment of the protein and CTAC
which has the conjugation site at the phenol, the overall light experienced by labels 3a–3c appears to be less polar based on
yield was observed to be minimally affected by conjugation to the observed increases in the light yields for the conjugates.
three different proteins (Fig. 6). The observed enhancements BSA in particular is known to bind a variety of ligands11 and
in light yield caused by CTAC for 4 and its protein conjugates acridinium ester conjugation at the acridinium nitrogen places
were also quite similar except for the neutral anti-HBsAg anti- the acridinium ring closer to the protein surface and more sus-
body as can be noted in Table 2. For example, CTAC enhance- ceptible to solubilization in less polar regions of the protein.
ments in the chemiluminescence of the free label 4 compared Because the fluorescence quantum yield of acridones actually
to its BSA, anti-TSH Mab and anti-HBsAg Mab conjugates were decreases with decreasing solvent polarity,1e,12 the mechanism
observed to be 4.1, 3.7, 4.6 and 6.1, respectively.
of light enhancement for the protein conjugates of 3a–3c must
For labels 3a–3c with conjugation sites at the acridinium also involve facile formation of dioxetane/dioxetanone precur-
nitrogen, unlike for 4, a significant increase in light yield was sors in the combined micellar/protein microenvironment and
observed upon protein conjugation (Fig. 6). For example for not because excited state acridone is constrained in a less
3a, conjugation to BSA, anti-TSH and anti-HBsAg antibodies polar environment on the protein.
was observed to result in an increase in light yield of 1.8, 2.0
Besides the increased specific activity, the increased stabi-
and 1.6-fold respectively compared to the free label (Fig. 6). lity of chemiluminescent acridinium esters is another useful
Similar increases were noted for labels 3b and 3c. Is this property for applications in commercial assays. Poor stability
increase in light yield in the protein conjugates of 3a–3c due can compromise assay sensitivity and decrease reagent shelf
to greater surfactant-mediated enhancement? The enhance- life. The acridinium esters 3a–3c showed increased light
ments in light yields for 2a/3a and its protein conjugates output when conjugated to proteins which is very attractive for
caused by CTAC were observed to be 4.3, 4.8, 5.9 and 6.1-fold improving the immunoassay sensitivity. We also compared the
for the free label versus its BSA, anti-TSH and anti-HBsAg anti- chemiluminescence stability of the BSA conjugates of these
body conjugates, respectively. Thus, even though a large labels with that of the conventional label 4 at two different pH
increase (1.8-fold) in light yield was observed for the BSA con- values and the results are shown in Tables 3 and 4. As can be
jugate of 3a (Fig. 6), this increase cannot be attributed solely noted from these tables, BSA conjugates of 3a–3c were
to greater CTAC enhancement in the conjugate versus the free observed to be equally stable as the conjugate of 4 at both pH
label (1.1-fold, Table 2). Similarly, although greater CTAC = 6 and 7.4 showing only a small drop of approximately 10%
enhancements were observed for the anti-TSH and anti- in activity after 30 days at room temperature. Reagents on auto-
HBsAg antibody conjugates of 3a, the magnitude of these mated instruments such as the ADVIA Centaur® systems are
enhancements (1.4-fold) are not sufficient to explain the kept refrigerated so the loss of chemiluminescence under
observed increases (2.0-fold for anti-TSH Mab and 1.6-fold for these conditions is expected to be minimal based on the stabi-
anti-HBsAg Mab) in light yields in Fig. 6 based solely on lity data at room temperature.
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10 micron, 3.9 × 300 mm column were used with MeCN–water
(each with 0.5% trifluoroacetic acid, TFA) as the solvents at a
flow rate of 1 mL minute−1 and UV detection at 260 nm. Pre-
parative HPLC was performed using a Phenomenex, Luna C18,
5 micron, 30 × 250 mm column at a flow rate of 20 mL
minute−1 and UV detection at 260 nm. MALDI-TOF (Matrix-
Assisted Laser Desorption Ionization-Time of Flight) mass
spectroscopy was performed using a VOYAGER DE Biospectro-
metry Workstation from ABI. This is a bench top instrument
operating in the linear mode with a 1.2 meter ion path length,
flight tube. Spectra were acquired in positive ion mode. For
small molecules, α-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 μL) ElectroSpray Ionization
(ESI) on a Waters Qtof API US instrument in the positive
ion mode. Optimized conditions are as follows: capillary =
3000 kV, cone = 35, source temperature = 120 °C, desolvation
temperature = 350 °C.
Table 3 Percent residual chemiluminescence (average of five repli-
cates) of BSA conjugates of 3a–3c and 4 in phosphate buffer, pH 6 at
25 °C. (CV = coefficient of variation of measurements)
Day
4 (%CV)
3a (%CV)
3b (%CV)
3c (%CV)
1
8
17
23
30
100 (0.3)
100 (2.3)
98 (3.2)
94 (2.8)
88 (1.2)
100 (2.6)
100 (2.5)
99 (2.1)
94 (1.1)
90 (0.9)
100 (2.8)
101 (1.8)
98 (2.2)
92 (0.3)
93 (2.4)
100 (4.8)
99 (3.9)
98 (6.5)
91 (4.5)
87 (3.3)
Table 4 Percent residual chemiluminescence (average of five repli-
cates) of BSA conjugates of 3a–3c and 4 in phosphate buffer, pH 7.4
at 25 °C
Day
4 (%CV)
3a (%CV)
3b (%CV)
3c (%CV)
1
8
17
23
30
100 (2.8)
96 (2.3)
96 (3.8)
89 (1.8)
89 (2.1)
100 (1.0)
98 (3.6)
96 (3.3)
95 (4.1)
92 (2.7)
100 (1.8)
94 (3.3)
94 (2.6)
89 (3.2)
89 (3.6)
100 (2.5)
97 (1.1)
95 (2.9)
91 (2.7)
87 (2.7)
Conclusions
NMR spectra (proton and carbon) were recorded on a
Bruker 600 MHz spectrometer. To suppress pseudobase for-
mation, spectra of the acridinium esters and intermediates
were acquired in an acidic solvent, namely deuterated tri-
fluoroacetic acid so that two forms of the compounds were
not present during spectral analysis. Chemiluminescence
measurements were carried out using a Berthold Technologies’
AutoLumat Plus LB 953 luminometer. Emission spectra were
recorded in >90% aqueous media using a PR-740 spectroradio-
meter (camera) from Photo Research Inc. Bandwidth slit:
2 nm; aperture: 0.5–2 degrees; exposure time: 5000 ms.
We have described the syntheses of three new acridinium
esters with conjugation sites at the acridinium nitrogen using
a simple and general synthetic protocol starting from easily
synthesized N-sulfopropylacridinium esters. In this study, we
have provided the first direct comparison of the properties of
chemiluminescent acridinium dimethylphenyl esters with
different conjugation sites, one where the light emitting acri-
done moiety is cleaved from its conjugate partner (convention-
al labeling) and another where the acridone remains attached
to the conjugate. Our results show that the conjugation at the
acridinium nitrogen, which also alters how the acridinium
ring and phenol are oriented with respect to the protein
surface, has a minimal impact on emission kinetics and emis-
sion spectra. However, this mode of conjugation to three
different proteins was observed to significantly increase
light yield which should be useful for improving the immu-
noassay sensitivity. How orientation of the acridinium ester
molecule on the protein surface affects non-specific binding
and the actual immunoassay performance are areas for further
investigation.
Synthesis of acridinium esters (Fig. 3)
Compound 2a. A solution of compound
1 (50 mg,
0.07 mmole) in 1 : 1 DMF–MeCN (6 mL) was treated with com-
pound i (Aldrich, 0.051 mL, 5 equivalents). The reaction was
heated at 75 °C in an oil bath under a nitrogen atmosphere for
1 hour. HPLC analysis using a Phenomenex, C18, 10 micron,
3.9 × 300 mm column and a 30 minute gradient of 10 → 100%
MeCN–water (each with 0.05% TFA) at a flow rate of 1 mL
minute−1 and UV detection at 260 nm showed clean conver-
sion to product 2a eluting at 16.5 minutes. The product was
purified by preparative HPLC using the same gradient and the
HPLC fractions were frozen at −80 °C and lyophilized to
dryness. Yield = 34 mg (71%). 1H-NMR (CF3COOD) 1.58 (d, 6H,
J = 6.1 Hz), 2.61 (s, 6H), 2.92 (m, 2H), 3.57 (brt, 2H), 3.62 (brt,
Experimental
General
Chemicals were purchased from Sigma-Aldrich (Milwaukee, 2H), 3.81 (brt, 2H), 3.93–4.03 (m, 8H), 4.14 (s, 3H), 4.98 (spt,
Wisconsin, USA) unless indicated otherwise. All the final acri- 1H, J = 6.1 Hz), 5.77 (brt, 2H), 7.94 (d, 1H, J = 2.6 Hz), 8.06 (s,
dinium esters, intermediates and reaction mixtures were ana- 2H), 8.16 (dd, 1H, J = 8.8, 6.8 Hz), 8.26 (m, 1H), 8.50 (m, 1H),
lyzed and/or purified by HPLC using a Beckman-Coulter HPLC 8.72 (brd, 2H), 8.79 (d, 1H, J = 8.8 Hz); 13C-NMR (CF3COOD)
system. For analytical HPLC, either a Phenomenex, Kinetex, 18.55, 18.76, 22.20, 22.23, 24.84, 42.77, 44.43, 51.68, 51.85,
C18, 2.6 micron, 4.6 × 50 mm column or a Phenomenex C18, 55.11, 67.98, 71.79, 72.17, 75.28, 75.40, 107.16, 119.88, 121.63,
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126.27, 128.22, 130.06, 130.72, 131.86, 133.23, 133.50, 138.90, eluting at 7.3 minutes. The product was purified by preparative
140.45, 141.14, 141.53, 147.65, 154.26, 160.98, 165.79, 172.25; HPLC using a Phenomenex, Luna C18, 5 micron, 30 × 250 mm
MALDI-TOF MS m/z 696.9 M+; HRMS m/z 696.2964 M+ column and a 30 minute gradient of 10 → 90% MeCN–water
(696.2955 calculated).
(each with 0.05% TFA) at a flow rate of 20 mL minute−1 and
Compound 2b. A solution of compound
1
(50 mg, UV detection at 260 nm. HPLC fractions containing the
0.07 mmole) in 1 : 1 DMF–MeCN (6 mL) was treated with com- product were frozen at −80 °C and lyophilized to dryness.
pound ii (0.1 g, 5 equivalents). The reaction was heated at Yield = 6.4 mg (61%). 1H-NMR (CF3COOD) 1.54 (d, 6H, J =
75 °C in an oil bath under a nitrogen atmosphere for 1 hour. 6.1 Hz), 2.25 (m, 2H), 2.57 (s, 6H), 2.77 (brt, 2H), 2.84 (brt,
HPLC analysis as described above showed clean conversion to 2H), 2.86–2.94 (m, 2H), 3.06 (s, 4H), 3.59 (t, 2H, J = 5.1 Hz),
product 2b eluting at 17.0 minutes. The product was purified 3.78 (m, 4H), 3.90–4.00 (m, 8H), 4.10 (s, 3H), 4.94 (spt, 1H,
by preparative HPLC using the same gradient and the HPLC J = Hz), 5.75 (brt, 2H), 7.90 (d, 1H, J = 2.6 Hz), 8.02 (s, 2H), 8.12
fractions were frozen at −80 °C and lyophilized to dryness. (m, 1H), 8.23 (m, 1H), 8.46 (m, 1H), 8.70 (brd, 2H), 8.75 (brd,
Yield = 36 mg (64%). 1H-NMR (CF3COOD) 1.59 (d, 6H, J = 1H); 13C-NMR (CF3COOD) 18.52, 18.72, 22.16, 22.19, 24.78,
6.1 Hz), 2.62 (s, 6H), 2.95 (m, 2H), 3.56 (brt, 2H), 3.64 (brt, 26.90, 27.24, 31.43, 35.61, 42.93, 44.40, 51.62, 51.84, 70.77,
2H), 3.84 (brt, 2H), 3.97 (m, 18H), 4.03 (m, 2H), 4.15 (s, 3H), 71.84, 72.33, 75.24, 75.36, 107.12, 119.85, 121.59, 126.23,
4.99 (spt, 1H, J = 6.1 Hz), 5.80 (brt, 2H), 7.95 (d, 1H J = 2.5 Hz), 127.28, 128.18, 130.01, 130.69, 131.82, 133.18, 133.46, 138.85,
8.08 (s, 2H), 8.17 (dd, 1H, J = 8.8, 6.8 Hz), 8.28 (dd, 1H, J = 140.43, 141.08, 141.50, 147.59, 154.20, 160.94, 165.76, 171.13,
9.9, 2.6 Hz), 8.48–8.54 (m, 1H), 8.75 (brd, 2H), 8.80 (d, 1H, J = 172.22, 176.15, 178.98; MALDI-TOF MS m/z 907.1 M+; HRMS
8.8); 13C-NMR (CF3COOD) 18.55, 18.75, 22.20, 22.22, 24.82, m/z 907.3459 M+ (907.3435 calculated).
42.52, 44.45, 51.63, 51.87, 55.08, 68.21, 71.74, 71.82, 72.10,
Compound 3b. A solution of compound 2b (10 mg,
72.36, 75.26, 75.39, 107.15, 119.90, 121.65, 126.26, 128.20, 0.012 mmole) in acetonitrile (2.5 mL) was mixed with 0.1 M
130.04, 130.71, 131.85, 133.22, 133.49, 138.87, 140.46, 141.11, sodium phosphate pH = 7.2 (1 mL) and DSG (39.5 mg,
141.54, 147.60, 154.24, 160.96, 165.80, 172.26; MALDI-TOF 10 equivalents) was added. The reaction was stirred at room
MS m/z 828.9 M+; HRMS m/z 828.3731 M+ (828.3741 temperature for 1 hour. HPLC analysis as described above
calculated).
showed clean conversion to product 3b eluting at 7.3 minutes.
Compound 2c. A solution of compound
1
(50 mg, The product was purified by preparative HPLC using a
0.07 mmole) in DMSO (6 mL) was treated with compound iii 30 minute gradient of 10 → 70% MeCN–water (each with
(0.15 g, HBr salt, 5 equivalents) and diisopropylethylamine 0.05% TFA) and the HPLC fraction was frozen at −80 °C and
(0.12 mL, 10 equivalents). The reaction was heated at 75 °C in lyophilized to dryness. Yield
=
6.3 mg (50%). 1H-NMR
an oil bath under a nitrogen atmosphere for 1 hour. HPLC (CF3COOD) 1.55 (d, 6H, J = 6.1 Hz), 2.26 (m, 2H), 2.58 (s, 6H),
analysis as described above showed clean conversion to 2.80 (brt, 2H), 2.86 (brt, 2H), 2.88–2.95 (m, 2H), 3.08 (s, 4H),
product 2b eluting at 15.0 minutes. The product was purified 3.60 (brt, 2H), 3.70–3.80 (m, 4H), 3.90–4.00 (m, 20H), 4.11
by preparative HPLC using the same gradient and the HPLC (s, 3H), 4.95 (spt, 1H, J = 6.1 Hz), 5.76 (brt, 2H), 7.91 (d, 1H, J =
fractions were frozen at −80 °C and lyophilized to dryness. 2.3 Hz), 8.04 (s, 2H), 8.13 (m, 1H), 8.24 (m, 1H), 8.48 (m, 1H),
Yield = 29 mg (52%). 1H-NMR (CF3COOD) 1.61 (d, 6H, J = 6.1 Hz), 8.71 (brd, 2H), 8.77 (d, 1H J = 8.6 Hz); 13C-NMR (CF3COOD)
2.35 (m, 2H), 2.64 (m, 10H), 2.95 (m, 2H), 3.30 (s, 3H), 18.54, 18.74, 22.19, 22.23, 22.43, 24.83, 26.94, 27.26, 31.43,
3.50 (brt, 2H), 3.54 (brt, 2H), 3.58 (brt, 2H), 3.68–3.79 (m, 4H), 35.41, 35.46, 43.24, 44.42, 51.66, 51.87, 55.16, 71.60, 71.88,
3.83 (brt, 2H), 3.85–3.91 (m, 2H), 4.17 (s, 3 H), 5.00 (spt, 1H, 72.10, 72.42, 75.27, 75.38, 107.14, 119.89, 121.63, 126.26,
J = 6.1 Hz), 5.80 (brt, 2H), 7.97 (d, 1H, J = 2.5 Hz), 8.09 (s, 2H), 128.20, 130.04, 130.72, 131.85, 133.21, 133.49, 133.88, 140.46,
8.19 (dd, 1 H, J = 8.8, 6.8 Hz), 8.30 (m, 1H), 8.53 (m, 1H), 8.74 141.11, 141.53, 147.61, 154.22, 160.96, 165.78, 171.08, 172.24,
(brd, 2H), 8.82 (d, 1H, J = 8.7 Hz); 13C-NMR (CF3COOD) 18.55, 176.18, 179.00; MALDI-TOF MS m/z 1039.2 M+; HRMS m/z
18.76, 20.07, 22.20, 22.23, 22.67, 24.76, 25.32, 40.16, 42.06, 1039.4260 M+ (1039.4222 calculated).
49.76, 51.36, 51.83, 55.10, 61.46, 62.48, 62.68, 75.29, 75.41,
Compound 3c. A solution of compound 2c (9 mg,
107.23, 119.85, 121.58, 126.27, 128.21, 130.06, 130.72, 131.87, 0.011 mmole) in acetonitrile (1.5 mL) was mixed with 0.1 M
133.23, 133.50, 138.89, 140.43, 141.18, 141.53, 147.65, 154.24, sodium phosphate pH = 7.2 (1 mL) and DSG (40 mg, 11 equiva-
160.99, 165.77, 172.25; MALDI-TOF MS m/z 816.1 M+; HRMS lents) was added. The reaction was stirred at room temperature
m/z 815.3387 M+ (815.3360 calculated).
for 1 hour. HPLC analysis as described above showed 80% con-
Compound 3a. A solution of compound 2a (8 mg, version to product 3c eluting at 6.4 minutes. The product was
0.011 mmole) in acetonitrile (1 mL) was mixed with 0.1 M purified by preparative HPLC using a 40 minute gradient of 10
sodium phosphate pH = 7.2 (1 mL) and disuccinimidyl gluta- → 60% MeCN–water (each with 0.05% TFA) and the HPLC frac-
rate (DSG, 37.5 mg, 10 equivalents) was added. The reaction tion was frozen at −80 °C and lyophilized to dryness. Yield =
was stirred at room temperature for 1 hour. HPLC analysis 7.5 mg (66%). 1H-NMR (CF3COOD) 1.55 (d, 6H, J = 6.1 Hz),
using a Phenomenex, Kinetex C18, 2.6 micron, 4.6 × 50 mm 2.21 (m, 2H), 2.24–2.34 (m, 4H), 2.53–2.60 (m, 8H), 2.65
column and a 10 minute gradient of 10 → 90% MeCN–water (m, 2H), 2.82 (t, 2H, J = 6.8 Hz), 2.89 (m, 2H), 3.08 (s, 4H), 3.21
(each with 0.05% TFA) at a flow rate of 1 mL minute−1 and UV (s, 3H), 3.44 (m, 2H), 3.48–3.56 (m, 4H), 3.58 (brt, 2H),
detection at 260 nm showed clean conversion to product 3a 3.61–3.71 (m, 2H), 3.77 (m, 4H), 4.11 (s, 3H), 4.95 (spt, 1H, J =
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6.0 Hz), 5.75 (brt, 2H), 7.91 (d, 1H, J = 2.6 Hz), 8.03 (s, 2H), bottom of a cuvette. Cuvettes were placed into the primed
8.13 (m, 1H), 8.25 (m, 1H), 8.48 (m, 1H), 8.69 (brd, 2H), LB953 and the chemiluminescence reaction was initiated with
8.76 (brd, 1H); 13C-NMR (CF3COOD) 18.46, 18.66, 22.11, the sequential addition of 0.3 mL of reagent 1, a solution of
22.14, 22.40, 26.86, 27.20, 31.46, 39.45, 42.04, 49.67, 0.5% hydrogen peroxide in 0.1 M nitric acid followed by the
50.37, 50.71, 51.33, 51.75, 55.01, 61.98, 62.26, 75.20, 75.31, addition of 0.3 mL of reagent 2, a solution of 0.25 M sodium
107.12, 119.77, 121.51, 126.17, 128.12, 129.96, 130.63, 131.78, hydroxide with or without a surfactant. For measurements
133.13, 133.40, 138.81, 140.35, 141.09, 141.44, 147.56, 154.15, with CTAC, 7 mM of this cationic surfactant was included in
160.89, 165.69, 171.17, 172.16, 176.16, 178.92; MALDI-TOF reagent 2.
MS m/z 1026.6 M+; HRMS m/z 1026.3817 M+ (1026.3840
calculated).
Each chemiluminescence flash curve was measured in 100
intervals of 0.1 second (10 seconds total time, CTAC) or 240
intervals of 0.5 seconds (2 minutes total time, no surfactant)
from the point of chemiluminescence initiation with the
Synthesis of protein conjugates of acridinium esters (Table 1)
The following procedure illustrated for labeling of BSA was addition of 0.25 M NaOH. Each chemiluminescence reaction
used for labeling the anti-TSH and anti-HBsAg antibodies as was carried out a minimum of three times, averaged and
well.
converted to a percentage of the chemiluminescence accumu-
BSA (1 mg, 15 nanomoles, 0.2 mL of a 5 mg mL−1 solution) lated up to each time interval. The output from the lumin-
was diluted with 0.2 mL of 0.1 M sodium carbonate, pH 9. The ometer instrument was expressed as R.L.Us (Relative Light
protein solutions, in separate labeling reactions, were treated Units).
with ten equivalents of various acridinium esters 3a–3c and 4.
Emission wavelength measurements (Fig. 5, S11 and S12)
The acridinium esters were dissolved in dimethyl sulfoxide to
give 5 mg mL−1 solutions. For labeling with the various acridi-
Visible wavelength emission spectra of the acridinium esters 4
nium esters, 0.0302 mL of 4, 0.0274 mL of 3a, 0.0314 mL of 3b
and 0.0309 mL of 3c were added. The reactions were stirred at
room temperature for 2–3 hours. The reactions were then
diluted with 0.5 mL de-ionized water and transferred to 4 mL
Amicon filters from Millipore (MW 30 000 cutoff). The labeling
reactions in the filters were further diluted with 3 mL de-
ionized water. The solutions were concentrated to ∼0.1 mL by
centrifugation at 3500g for 10 minutes. This dilution and con-
centration process was repeated three more times. The final
conjugate solutions were transferred to vials and the solutions
were frozen and lyophilized. The lyophilized conjugates were
dissolved in de-ionized water to give ∼2 mg mL−1 solutions
(protein assay). The conjugates 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 measurements. By knowing the molecular weight of the
specific acridinium ester label, the extent of label incorpor-
ation for that specific acridinium ester could thus be calcu-
lated (Table 1).
(amine derivative), 2a–2c and the protein conjugates of 4, 3a–
3c were measured by FSSS (Fast Spectral Scanning System)
using
a PR-740 spectroradiometer (camera) from Photo
Research Inc. The following instrument parameters were used:
bandwidth slit: 2 nm; aperture: 0.5–2 degrees; exposure time:
5000 ms. In a typical measurement, 0.01 mL of a 5 mg mL−1
solution of the acridinium ester in DMSO or 0.025–0.05 mL of
the protein conjugates at 2 mg mL−1 were diluted with 0.3 mL
of reagent 1 comprising 0.5% hydrogen peroxide in 0.1 M
nitric acid. Just prior to the addition of reagent 2 comprising
7 mM CTAC in 0.25 M sodium hydroxide, the shutter of the
camera was opened and light was collected for 5 seconds. The
output of the instrument is a graph of light intensity versus
wavelength.
Chemiluminescence stability measurements (Tables 3 and 4)
The acridinium ester labeled BSA conjugates were diluted to a
concentration of 0.2 nM in an aqueous buffer of 10 mM phos-
phate, 0.15 M NaCl, 8 mM sodium azide and 0.015 mM bovine
serum albumin (BSA), pH = 6.0 or 7.4. Two milliliter volumes
of the diluted conjugates were sealed in glass vials and incu-
bated at 25 °C for 30 days protected from light. Chemilumines-
cence of 0.01 mL samples (five replicates) of the diluted
conjugates was periodically measured as a function of time.
Chemiluminescence was measured on a Berthold Techno-
logies’ AutoLumat Plus LB953 luminometer. The averaged
results were calculated as residual chemiluminescence percen-
tages with respect to values determined at day 1.
Chemiluminescence measurements (Fig. 4, 6, S7–S10 ESI†)
The chemiluminescence of free labels and protein conjugates
of acridinium esters (Fig. 3) was measured on an Autolumat
LB953 Plus luminometer from Berthold Technologies. Free
labels, amine derivative of 4 and 2a–2c, were dissolved in
DMSO to give 0.2–0.5 mM solutions. These solutions were seri-
ally diluted 106-fold for chemiluminescence measurements in
an aqueous buffer of 10 mM disodium hydrogen phosphate,
0.15 M NaCl, 8 mM sodium azide and 0.015 mM bovine serum
Acknowledgements
albumin (BSA), pH = 8.0. Protein conjugates, ∼2 mg mL−1
,
were serially diluted 105-fold for chemiluminescence measure- We thank Mr Richard Wright for NMR spectra and
ments in the same buffer. A 0.010 mL volume of each diluted Dr Shenliang Wang for assistance with emission spectrum
acridinium ester or conjugate sample was dispensed into the measurements.
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