Regiodependent Luminescence Quenching of Biotinylated N-Sulfonyl- acridinium-9-carboxamides by Avidin

Article abstract of DOI:10.1021/ol035323p

(Matrix presented) Biotin was conjugated to chemiluminescent N-sulfonylacridinium-9-carboxamides at the N-10 or 9-position carboxamide. Upon binding to avidin, the light output of the N-10 derivative (8) was quenched up to 92% upon triggering with basic p

Full text of DOI:10.1021/ol035323p

ORGANIC
LETTERS
2003
Vol. 5, No. 21
3779-3782
Regiodependent Luminescence
Quenching of Biotinylated
N-Sulfonyl-acridinium-9-carboxamides by
Avidin
Maciej Adamczyk,* Phillip G. Mattingly, Jeffrey A. Moore, and You Pan
Department of Chemistry (D09MD), Diagnostics DiVision, Abbott Laboratories,
100 Abbott Park Road, AP20, Abbott Park, Illinois 60064-6016
maciej.adamczyk@abbott.com
Received July 17, 2003
ABSTRACT
Biotin was conjugated to chemiluminescent N-sulfonylacridinium-9-carboxamides at the N-10 or 9-position carboxamide. Upon binding to
avidin, the light output of the N-10 derivative (8) was quenched up to 92% upon triggering with basic peroxide, while the 9-position carboxamide
conjugate (9) was quenched only 33%. The utility of this effect was demonstrated in a model homogeneous chemiluminescence assay.
Chemiluminescence provides the most sensitive nonisotopic
detection technology available today. One class of com-
pounds extensively utilized in chemiluminescent detection
formats are the ester and N-sulfonylamide derivatives of
acridinium-9-carboxylic acid.1-3 Acridinium salts are highly
desirable as labels for detection due in part to a low naturally
occurring background signal in biological samples and the
relatively high quantum yields of the resultant chemi-
luminescent reaction. Such properties permit detection of
acridinium-labeled species over 5 orders of magnitude
typically starting at attomole levels.
derivatives have been prepared in efforts to enhance the
chemiluminescence quantum yield, to improve the stability
or solubility of the labels, and to modulate the kinetics or
color of the light generated in the chemiluminescent
reaction.2,4-8 Historically, these acridinium salt derivatives
have been synthesized with reactive functionalities incorpo-
rated into the ester or N-sulfonylamide moiety of the labels
for coupling to ligands of interest. Synthesis of labels with
reactive functionalities for coupling to ligands via other
positions on the acridinium moiety have been explored less
frequently.8-12
Numerous acridinium salt derivatives have been synthe-
sized and evaluated as labels for detection. Acridinium salt
(4) McCapra, F. Acc. Chem. Res. 1976, 9, 201-208.
(5) Kinkel, T.; Lubbers, H.; Schmidt, E.; Molz, P.; Skrzipczyk, H. J. J.
Biolumin. Chemilumin. 1989, 4, 136-139.
(1) Adamczyk, M.; Mattingly, P. G. In Luminescence Biotechnology:
Instruments and Applications; Van Dyke, K., Van Dyke, C., Woodfork,
K., Ed.; CRC Press: Washington, DC, 2002; p 77-105.
(2) Pringle, M. J. J. Clin. Ligand Assay 1999, 22, 105-121.
(3) Handley, R. S.; Akhavan-Tafti, H.; Schaap, A. P. J. Clin. Ligand
Assay 1997, 20, 302-312.
(6) Mattingly, P. G. J. Biolumin. Chemilumin. 1991, 6, 107-114.
(7) Adamczyk, M.; Chen, Y.-Y.; Mattingly, P. G.; Moore, J. A.; Shreder,
K. Tetrahedron 1999, 55, 10899-10914.
(8) Renotte, R.; Sarlet, G.; Thunus, L.; Lejeune, R. Luminescence 2000,
15, 311-320.
10.1021/ol035323p CCC: $25.00 © 2003 American Chemical Society
Published on Web 09/16/2003

The general mechanism for acridinium salt chemilumi-
nescence is depicted in Scheme 1.7,13 Chemiluminescence
emitting species is dissociated from potential collisional
quenching entities (i.e., proteins). In contrast, a derivative
linked to ligands of interest through the acridinium ring
results in generation of an excited-state acridone that remains
tethered to the ligand upon chemiluminescence triggering.
Tethered acridinium light-emitting species have the potential
to undergo collisional quenching or to serve as donors in
energy transfer processes.2
Scheme 1. Proposed Mechanism of Acridinium Salt
Chemiluminescence
Nonacridinium salt-based reagents have been described
in luminescence assays that rely on quenching or energy
transfer. Specifically, assays for biotin have been developed
utilizing the bioluminescent photoprotein aequorin.14-19 Bio-
luminescence resonance energy transfer (BRET) has recently
been demonstrated to be useful for studies of protein-protein
interactions.20-22 Similarly, chemiluminescence resonance
energy transfer assays utilizing aminobutylethylisoluminol
(ABEI) as the luminescent donor and fluorescein as the
acceptor have been successfully developed for several
biological ligands of interest.23-27
Here we describe the synthesis of an N10-carboxyalkyl-
functionalized acridinium label. The N10-carboxyalkyl acri-
dinium label was prepared for direct comparison to an
analogous acridinium label with the reactive functionality
for coupling to ligands of interest incorporated into the
sulfonamide moiety. The labels were coupled to a derivative
of biotin, generating model tethered and releasable acridone-
generating tracers. The resulting biotin tracers were evaluated
in terms of their chemiluminescent properties in buffer alone
and in the presence of the binding protein avidin.
is triggered by the addition of hydrogen peroxide anion to
the 9-position of the acridinium salt, yielding an acridan
hydroperoxide. Subsequent deprotonation of the hydro-
peroxide and attack at the carbonyl carbon provides a
tetrahedral spirodioxetane intermediate. Decomposition of
the unstable spirodioxetane intermediate results in formation
of an excited-state acridone with concomitant elimination
of CO2 and a leaving group X. Light is generated upon return
of the excited-state acridone to the ground state.
The mechanism in Scheme 1 illustrates that the site of
ligand attachment to an acridinium salt may impart a subtle
but potentially very important characteristic to the label.
Acridinium salt derivatives linked to ligands of interest
through the ester or sulfonamide moiety result in generation
of an excited-state acridone that is released upon triggering
of the chemiluminescent reaction (Scheme 2). From a
sensitivity standpoint, such a process is desirable, as the light-
The N10-carboxyalkyl-functionalized acridinium-9-carbox-
amide label 6 that results in a tethered acridone species upon
chemiluminescence triggering was synthesized as depicted
(9) Zomer, G.; Staventuiter, J. F. C. Anal. Chim. Acta 1989, 227, 11-
19.
(10) Sato, N. Tetrahedron Lett. 1996, 37, 8519-8522.
(11) Razavi, Z.; McCapra, F. Luminescence 2000, 15, 239-244.
(12) Mattingly, P. G.; Bennett, L. U.S. Pat. 5468646, 1995.
(13) Rak, J.; Skurski, P.; Blazejowski, J. J. Org. Chem. 1999, 64, 3002-
3008.
(14) Adamczyk, M.; Moore, J. A.; Shreder, K. Org. Lett. 2001, 3, 1797-
1800.
(15) Witkowski, A.; Ramanathan, S.; Daunert, S. Anal. Chem. 1994, 66,
1837-1840.
Scheme 2
(16) Crofcheck, C. L.; Grosvenor, A. L.; Anderson, K. W.; Lumpp, J.
K.; Scott, D. L.; Daunert, S. Anal. Chem. 1997, 69, 4768-4772.
(17) Feltus, A.; Grosvenor, A. L.; Conover, R. C.; Anderson, K. W.;
Daunert, S. Anal. Chem. 2001, 73, 1403-1407.
(18) Grosvenor, A. L.; Feltus, A.; Conover, R. C.; Daunert, S.; Anderson,
K. W. Anal. Chem. 2000, 72, 2590-2594.
(19) Gorokhovatsky, A. Y.; Rudenko, N. V.; Marchenkov, V. V.;
Skosyrev, V. S.; Arzhanov, M. A.; Burkhardt, N.; Zakharov, M. V.;
Semisotnov, G. V.; Vinokurov, L. M.; Alakhov, Y. B. Anal. Biochem. 2003,
313, 68-75.
(20) Boute, N.; Jockers, R.; Issad, T. Trends Pharmacol. Sci. 2002, 23,
351-354.
(21) Angers, S.; Salahpour, A.; Joly, E.; Hilairet, S.; Chelsky, D.; Dennis,
M.; Bouvier, M. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 3684-3689.
(22) Xu, Y.; Piston, D. W.; Hirschie Johnson, C. Proc. Natl. Acad. Sci.
U.S.A. 1999, 96, 151-156.
(23) Patel, A.; McCapra, F.; Davies, C. J.; Campbell, A. K. Biochem.
Soc. Trans. 1983, 11, 196-197.
(24) Patel, A.; Davies, C. J.; Campbell, A. K.; McCapra, F. Anal.
Biochem. 1983, 129, 162-169.
(25) Patel, A.; Campbell, A. K. Clin. Chem. 1983, 29, 1604-1608.
(26) Campbell, A. K.; Patel, A. Biochem. J. 1983, 216, 185-194.
(27) Williams, E. J.; Campbell, A. K. Anal. Biochem. 1986, 155, 249-
255.
3780
Org. Lett., Vol. 5, No. 21, 2003

1. The tethered acridone-generating tracer 8 was synthesized
by coupling of active ester 7 to biotin-PEO-amine. Similarly,
the dissociated acridone-generating tracer 9 was from biotin-
PEO-amine and the previously described sulfonamide-
derivatized acridinium active ester.28
Scheme 3
The biotin tracers 8 and 9 were directly compared in terms
of their chemiluminescence profiles and light output in assay
buffer (10 mM borate, 2 mM EDTA, 0.1% Tween-20, pH
7.3). Both tracers exhibited flash-type luminescence profiles
with all light being generated within 5 s after addition of
the chemiluminescence trigger (Figure 2). The overall
in Scheme 3. Specifically, sulfonamide 2 was prepared in
89% yield from 3-aminopropylsulfonate derivative 1 and
p-toluenesulfonyl chloride in THF. Acylation of the sulfon-
amide anion with acridinium-9-carboxylic acid chloride gave
the protected N-sulfonylacridine-9-carboxamide 4 in 40%
overall yield. Alkylation of the N-10 position on the acridine
ring with triflate-activated ethyl 10-hydroxydecanoate (5) and
acidic hydrolysis provided the desired acridinium-9-carbox-
amide label 6 in 87% yield. The N-hydroxysuccinimidyl
active ester of acridinium sulfonamide 6 was subsequently
prepared in g95% yield using N-succinimidyl trifluoro-
acetate. The sulfonamide-functionalized acridinium-9-
carboxamide label that results in release of an exited state
acridone species upon chemiluminescence triggering was
prepared following previously described procedures.28
The structures of the N-10 and 9-position biotin tracers
utilized in model studies described here are depicted in Figure
Figure 2. Representative luminescence profiles of the N-10-
tethered and 9-position biotin tracers. Luminescence profiles were
obtained by triggering 25 µL aliquots of tracer (∼0.6 nM) in 10
mM borate, 2 mM EDTA, 0.1% Tween-20, pH 7.3 using a
microplate luminometer (MicroLumat Plus, Perkin-Elmer). Chemi-
luminescence trigger solution: 200 µL 0.18 N NaOH, 0.7% H2O2,
1% Triton X-100, 0.05% diethylenetriaminepentacetic acid.
chemiluminescent output from the two tracers was also of
the same magnitude. The N-10 tracer yielded ∼2.3 × 1019
counts/mol. The 9-position tracer yielded ∼4.2 × 1019
counts/mol.
The tracers exhibited markedly different chemiluminescent
properties relative to each other in the presence of avidin.
Specifically, each biotinylated acridinium tracer (0.5 nM)
was individually mixed with several concentrations of avidin
(8-8000 pM) in assay buffer, and the solutions were
incubated for 45 min. A chemiluminescent response from
each mixture was subsequently obtained from 25 µL of each
mixture as described above. Plots of the normalized relative
light units (RLU) obtained versus avidin concentration are
shown in Figure 3. The data show that acridinium chemi-
luminescence from a biotin tracer can be quenched by avidin
in a concentration-dependent manner. Significantly, the
(28) Adamczyk, M.; Chen, Y.-Y.; Fino, J. R.; Mattingly, P. G. In
Proceeding of the XIth International Symposium on Bioluminescence and
Chemiluminescence; Case, J. F., Herring, P. J., Robison, B. H., Haddock,
S. H. D., Kricka, L. J., Stanley, P. E., Eds.; World Scientific: Asilomar,
CA, 2001; pp 207-210.
Figure 1. Structures of N-10-tethered and 9-position biotin tracers.
Org. Lett., Vol. 5, No. 21, 2003
3781

tures were subsequently added to a fixed concentration of
avidin also in assay buffer. The solutions were allowed to
incubate for 45 min, and 25 µL of each sample was triggered
as above.
The dose-response curve obtained is depicted in Figure
4. The average % quenching value reported in Figure 4 is
Figure 3. Plot of normalized chemiluminescent response for
tethered (8) (b) and releasable (9) biotin tracers (O) vs avidin
concentration. Data points represent the average of duplicate values.
chemiluminescent output derived from biotin tracer 8 result-
ing in generation of a tethered excited-state acridone upon
luminescent triggering is quenched up to ∼92% upon titration
with avidin. In contrast, biotin tracer 9, which results in
generation of a dissociated excited state acridone upon
luminescent triggering, is quenched only ∼33% upon titration
with avidin. Identical studies utilizing biotinylated acridini-
ums 8 and 9 and bovine serum albumin (BSA) were also
conducted as controls. Chemiluminescent outputs from these
controls over the entire BSA concentration range tested (8-
8000 pM) did not vary significantly (less than 1%). The
results demonstrate that the quenching phenomenon is due
to a specific binding interaction.
The avidin-specific modulation of acridinium chemilumi-
nescence was further demonstrated in a model homogeneous
assay. Thus, a fixed concentration of biotinylated tracer 8
(0.5 nM, tethered biotin) was added to several concentrations
of the water-soluble biotin derivative biotin-PEO-amine
(0.06-16 nM) in assay buffer. The biotin-containing mix-
Figure 4. Dose-response curve generated for biotin-PEO-amine.
Data points represent the average of triplicate values. The curve
represents the best nonlinear fit of the data using a four-parameter
logistic.
relative to the chemiluminescent response obtained for
biotinylated tracer 8 in buffer alone. As expected, acridinium
chemiluminescence quenching due to association of avidin
with the biotinylated tracer decreases with increasing con-
centrations of the biotin analyte.
Investigation of the generality of this chemiluminescence-
quenching phenomenon in other assay formats is ongoing.
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