Intrinsic factor-mediated modulation of cyanocobalamin-N-sulfonyl- acridinium-9-carboxamide chemiluminescence

Article abstract of DOI:10.1016/j.bmcl.2004.05.062

Two cyanocobalamin-N-sulfonyl-acridinium-9-carboxamides with linkage through the N¹⁰ or 9-position were prepared from B ₁₂-e-carboxylic acid. The noncovalent association of intrinsic factor with these ligands resulted in specific mod

Full text of DOI:10.1016/j.bmcl.2004.05.062

Bioorganic & Medicinal Chemistry Letters 14 (2004) 3917–3921
Intrinsic factor-mediated modulation of cyanocobalamin–
N-sulfonyl-acridinium-9-carboxamide chemiluminescence
Maciej Adamczyk,* Donald D. Johnson, Phillip G. Mattingly,
Jeffrey A. Moore and You Pan
Department of Chemistry (D09MD), Diagnostics Division, Abbott Laboratories, 100 Abbott Park Road, AP20,
Abbott Park, IL 60064-6016, USA
Received 13 April 2004; revised 21 May 2004; accepted 24 May 2004
Available online 19 June 2004
Abstract—Two cyanocobalamin–N-sulfonyl-acridinium-9-carboxamides with linkage through the N10 or 9-position were prepared
from B12-e-carboxylic acid. The noncovalent association of intrinsic factor with these ligands resulted in specific modulation of the
associated chemiluminescence signal either by quenching or changing the emission profile. Either effect was sufficient to formulate a
homogeneous assay to detect vitamin B12 in buffer.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Chemiluminescent N-sulfonyl-acridinium-9-carboxamide
reporter groups have been successfully commercialized
on high-throughput, automated in vitro diagnostic
analyzers that utilize a heterogeneous assay format, that
is, the bound and free labeled material was physically
separated before the signal is generated.1 We have re-
cently reported that homogeneous chemiluminescent
assays are possible using the same family of N-sulfonyl-
acridinium-9-carboxamide reporter groups. Using the
specific interaction of proteins as divergent as avidin and
folate binding protein (FBP) with their complementary
N-sulfonyl-acridinium-9-carboxamide-labeled ligands,
assays for biotin2 and folic acid3 were demonstrated.
Both assays relied on the quenching of the chemilumi-
nescence signal from the labeled ligand by the specific
binding protein, a phenomenon that was most efficient
when the N-sulfonyl-acridinium-9-carboxamide–ligand
linkage was via the N10-position on the acridinium nu-
cleus.
lation of the associated chemiluminescence signal either
by quenching or by a change in the chemiluminescence
emission profile. Either effect was sufficient to formulate
a homogeneous assay to detect vitamin B12.
In this report we demonstrate that a noncovalent pro-
tein–ligand interaction between chemiluminescent
N-sulfonyl-acridinium-9-carboxamide–cyanocobalamin
ligands and intrinsic factor can result in specific modu-
Cyanocobalamin (1, vitamin B12) is routinely monitored
for the diagnosis and clinical management of ailments
(e.g., anemia, atherosclerosis, and neuropsychiatric dis-
orders) arising from the impairment of the enzyme,
methionine synthase.4;5 Porcine intrinsic factor (IF), a
gut-secreted 58–64 kDa glycoprotein6;7 that binds B12
with high affinity (association constant 1.5–
3.3 · 1010 Mꢀ1),7;8 has been used successfully in serum
assays for B12.5;9–11
Keywords: Homogeneous chemiluminescent assays; Intrinsic factor;
Vitamin B12; Cobalamin.
* Corresponding author. Tel.: +1-847-937-0225; Fax: +1-847-938-89-
27; e-mail: maciej.adamczyk@abbott.com
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.05.062

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M. Adamczyk et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3917–3921
Scheme 1. Preparation of cyanocobalamin–N-sulfonyl-acridinium-9-carboxamide intrinsic factor ligands. Reagents and conditions: (a) 0.8 M aq
H3PO4, 70 ꢁC, 4 h, 10%; (b) H2N(CH2)6NH2, imidazole, EDAC, pH 4, 64%; (c) Et3N, DMF, overnight, 60%; (d) tosyl chloride, Et3N, 95%; (e) (i) K
t-OBu/THF; (ii) acridine-9-carbonylchloride, 10, 67%; (f) (i) TfO(CH2)9CO2CH3, 2,6-dibutyl-4-methylpyridine, CH2Cl2; (ii) aq HCl (1 N), reflux, 8 h;
(iii) CF3CO2C6F5, pyridine, CH2Cl2, 15%.
Thus, for this study two cyanocobalamin–N-sulfonyl-
acridinium-9-carboxamides with linkage through the
N10 or 9-position were prepared from B12-e-carboxylate
(Scheme 1). Cyanocobalamin (1) was partially hydro-
lyzed in dilute phosphoric acid to afford the e-carboxylic
acid 2, then converted to the 6-aminohexyl amide 3.10
The acridinium-9-carboxamide active esters 412 and 5
bearing linkers extending from the 9- and 10-positions,
respectively, were conjugated to cyanocobalamin 3 to
give the chemiluminescent cobalamin ligands for IF, 6
and 7.
integration period differed by an order of magnitude,
unlike the analogous biotin2 and folate3 conjugates
previously described. The integrated signal for com-
pound 6, where the cyanocobalamin was attached at the
9-position carboxamide, was 5.19 · 1019 RLU/mol, while
the signal from compound 7, with the label attached at
the N10-position,was only 4.9 · 1018 RLU/mol, that is,
the signal is quenched by over 90%. This result is con-
sistent with the mechanism of the chemiluminescent
reaction (Fig. 2), in which the acridone chemilumino-
phore is released on reaction with peroxide and base
from the cyanocobalamin in 6 but remains tethered in
compound 7. Analogous quenching of tethered fluoro-
phores by cyanocobalamin has been reported.13–17
The chemiluminescence of 6 and 7 was first evaluated in
the absence of IF. Serial dilutions of the two ligands
were prepared in buffer (10 mM borate, 2 mM EDTA,
0.1% Tween-20, pH 7.3) from initial stock solutions
(concentrations calculated based on the A550 nm for
vitamin B12, e550 ¼ 8739 Mꢀ1 cmꢀ1). As shown in Figure
1, signal generated from the two compounds over a 30 s
Upon examination of the chemiluminescence profile of
each ligand further differences were noted. The emission
from the 9-position carboxamide ligand 6 (Fig. 3a, red
trace) was noticeably faster (2 s), than the 10-position

M. Adamczyk et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3917–3921
3919
Figure 1. Chemiluminescence of ligands 6 and 7. Aliquots (50 lL) of
each ligand (10 mM borate, 2 mM EDTA, 0.1% Tween-20, pH 7.5)
were triggered (200 lL 0.18 N NaOH, 0.7% H2O2, 1% Triton X-100,
0.05% diethylenetriaminepentaacetic acid) in a microplate luminome-
ter (MicroLumat Plus, Perkin–Elmer). Data points represent the
average integrated RLUs of duplicate values obtained over a 30 s
window. The data was fit using linear regression analysis with pro-
portional weighting in Grafit (Erithacus Software).
Figure 2. Excited state acridones generated from 6 and 7.
and folic acid3 acridinium derivatives, equivalent flash
chemiluminescent profiles were observed regardless of
the point of attachment to the ligand.
ligand 7 (20 s) (Fig. 3b, red trace). The broader chemi-
luminescence profile exhibited by 7, was more typical of
acridinium-9-carboxamides that are sterically hindered
about the 9-position,18;19 or triggered under non-
optimum conditions (e.g. low peroxide anion concen-
tration). In the case of the N10 and 9-position biotin2
When examined in the presence of intrinsic factor
(5 · molar excess), the maximal intensity of the sig-
nal from both ligands 6 and 7 was diminished. The
(a)
(b)
(c)
(d)
Figure 3. Effect of added IF on the chemiluminescence of ligands 6 and 7. Aliquots (50 lL) of each ligand/IF solution (10 mM borate, 2 mM EDTA,
0.1% Tween-20, pH 7.5) were triggered (200 lL 0.18 N NaOH, 0.7% H2O2, 1% Triton X-100, 0.05% diethylenetriaminepentaacetic acid) in a mi-
croplate luminometer (MicroLumat Plus, Perkin–Elmer) and the signal recorded for 30 s. (a) Chemiluminescence profile of 6 w/wo IF. (b)
Chemiluminescence profile of 7 w/wo IF. (c) Plots of normalized chemiluminescent response for 6 [0.5 nM, 10 s ( ) and 2 s ( ) integration)] versus IF
ꢁ
d
(0.0024–2.5 nM). (d) Plots of normalized chemiluminescent response for 7 [2 nM, 10 s ( ) and 2 s ( ) integration)] versus IF (0.01–10 nM).
ꢁ
d

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M. Adamczyk et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3917–3921
added to several concentrations of vitamin B12 (0.01–
8 ng/mL). The mixtures were incubated with intrinsic
factor (0.5 nM) for 1 h. Twenty five-lL aliquots of each
solution were then triggered and the chemiluminescent
response from each sample was subsequently integrated
over a 2 s window and normalized relative to the inte-
grated value obtained for tracer alone in buffer. The
dose–response curve obtained is depicted in Figure 4.
In summary, the chemiluminescence efficiency of acrid-
inium-9-carboxamide–cyanocobalamin conjugates was
affected by the regiochemistry of the linkage between the
components. N10-acridinium-9-carboxamide–cyanoco-
balamin conjugate 7 was quenched by over 90% and the
chemiluminescence emission profile was broadened rel-
ative to the 9-position conjugate 6. The chemilumines-
cence signal from ligand 7 was further quenched upon
specific binding to intrinsic factor, while interaction of 6
with intrinsic factor resulted in a broadening of its
chemiluminescence emission profile. The IF–specific
modulation of the chemiluminescence signal from these
ligands, whether from quenching or from changes in the
emission profile, was sufficient to demonstrate a homo-
geneous assay to detect vitamin B12.
Figure 4. Dose–response curve generated for vitamin B12. Solutions of
B12, ligand 6 (0.5 nM) and IF (0.5 nM) in buffer (10 mM borate, 2 mM
EDTA, 0.1% Tween-20, pH 7.4) were incubated for 1 h. Aliquots
(25 lL) were then triggered [200 lL 0.18 N NaOH, 0.7% H2O2, 1%
Triton-X100, 0.05% diethylenetriaminepentaacetic acid, MicroLumat
Plus (Perkin–Elmer)]. The chemiluminescent response from each
sample was subsequently integrated over a 2 s window and normalized
relative to the integrated value obtained for 6 alone in buffer. Data
points represent the average of triplicate values. The curve represents
the best nonlinear fit of the data using a four-parameter logistic.
chemiluminescence emission profile of compound 6 (Fig.
3a, blue trace) was broadened, but the overall integrated
signal was undiminished. The chemiluminescence emis-
sion profile of 7 (Fig. 3b, blue trace) was unchanged, but
the overall integrated signal was further decreased.
We continue to explore other binding protein/ligand-
acridinium-9-carboxamide pairs to further define the
general scope of this technology.
The effects on the chemiluminescent response of ligands
6 and 7 upon binding to intrinsic factor were dose-
dependent. For the acridinium-derivatized ligand 6, the
effect was only apparent when the data from the first 2 s
of the emission were considered (Fig. 3c, ꢂ). Under these
conditions, the signal decreased by 32%. The concen-
tration discrimination disappeared when the signal
integration was extended to 10 s (Fig. 3c, ꢁ). In contrast,
the data integration window (2 or 10 s) had little effect
on the dose-dependent response of ligand 7 to intrinsic
factor; in either case, the chemiluminescence was quen-
ched by 33–44% (Fig. 3d) relative to the unbound li-
gand.
References and notes
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