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M. Adamczyk et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1324–1328
300000
250000
200000
150000
100000
50000
0
B
A
5% GHb "non-glycated Hb"
1
40% GHb "glycated Hb"
Kapp
0.8
0.6
0.4
0.2
0
7
211 µM
14 25 µM
25 2.1 µM
36 0.43 µM
1.95 3.90 7.80 15.6 31.2 62.5 125 250 500
0.01
0.1
1
10
100
1000 10000
Total Hb (µM)
Total Hb (µM)
Figure 4. Chemiluminescent signal attenuation by Ônon-glycatedÕ and ÔglycatedÕ hemoglobin. (A) Illustrative raw data using probe 14. (B) Apparent
affinity of probes 7, 14, 25, and 36. Conditions: non-glycated hemoglobin (ABT, Inc.) contained the normal physiological level of glycation (5%),
while the glycated samples contained a high level of modification (40%). Solutions containing the chemiluminescent boronic acids (0.5 nM) and
hemoglobin were prepared in buffer (10 mM HEPES, 2 mM EDTA, and 0.1% Tween 20, pH 7.3). Aliquots (25 lL) of each sample were triggered
(200 lL, 0.35 N NaOH, 1.32% H2O2, 0.09% DTPA, and 2% Triton X-100) and the chemiluminescent response from each sample was integrated over
a 2s window. Data points represent the average of triplicate values.
References and notes
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39, 2090.
7. James, T. D.; Shimori, H.; Takeuchi, M.; Shinkai, S.
Chem. Commun. 1996, 705.
Figure 5. Glycated hemoglobin dose–response curve with chemilumi-
8. Hartley, J. H.; James, T. D.; Ward, C. J. J. Chem. Soc.,
nescent boronic acid probe 25. Conditions: whole blood samples
Perkin Trans. 1 2000, 3155.
(Trimar) were treated with K3Fe(CN)6 (130 mol %) and diluted to a
9. James, T. D.; Shinkai, S. Top. Curr. Chem. 2002,
final concentration of 10 lM along with 25 (0.5 nM) in buffer (10 mM
218, 159.
HEPES, 2 mM EDTA, and 0.1% Tween 20, pH 7.4). Aliquots (25 lL)
10. Wang, W.; Gao, X.; Wang, B. Curr. Org. Chem. 2002, 6,
of each sample were triggered (200 lL, 0.35 N NaOH, 1.32% H2O2,
0.09% DTPA, and 2% Triton X-100.) The chemiluminescent response
from each sample was subsequently integrated over a 2s window.
1285.
11. Wulff, G. Pure Appl. Chem. 1982, 54, 2093.
12. All new compounds were purified by reverse-phase HPLC
to greater than 95% purity and yielded the expected m/z by
electrospray mass spectrometry.
13. Lebeau, L.; Oudet, P.; Mioskowski, C. Helv. Chim. Acta
1991, 74, 1697.
14. Hawkins, R. T.; Snyder, H. R. J. Am. Chem. Soc. 1960,
82, 3863.
15. Cooper, C. R.; James, T. D. J. Chem. Soc., Perkin Trans. 1
2000, 963.
16. Newkome, G. R.; Behera, R. K.; Moorefield, C. N.;
here represent the average of triplicate reads for tripli-
cate samples (9 data points/sample). The response over
the clinically relevant %HbA1c range of 5–12% showed
good linearity (R = 0.9902) and slope, thus demonstrat-
ing the foundation for a homogeneous assay for glycat-
ed hemoglobin.
Baker, G. R. J. Org. Chem. 1991, 56, 7162.
17. Blincko, S.; Edwards, R. Ann. Clin. Biochem. 1998,
35, 140.
Further studies to expand the utility of homogeneous
chemiluminescent assays are in progress.