Angewandte
Chemie
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Table 2: Comparison of IC50 of ChE inhibitors on human erythrocyte AChE and serum BChE obtained
with 5b and DTNB
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Inhibitor
Probe
IC50 [nm]
BChE
Selectivity for AChE[a]
AChE
donepazil
BW284C51
physostigmine
tacrine
5b
11.8Æ1.5
19Æ7
5260Æ290
446
216
341
2550
14.7
0.57
0.10
0.27
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Am. Chem. Soc. 2005, 127, 68 – 69.
[12] K. Shimada, K. Mitamura, J. Chro-
matogr. B 1994, 659, 227 – 241.
DTNB[b]
5b
4100Æ1800
19800Æ750
48000
58.0Æ5.4
DTNB[c]
5b
18.8
142.0Æ3.9
27.9Æ2.4
111.0Æ5.3
108Æ7
2090Æ42
16.0Æ2.9
11.5Æ1.2
29Æ2
DTNB[d]
5b
DTNB[b]
[a] Expressed as IC50 for BChE divided by IC50 for AChE. [b] See Ref. [2b]. [c] See Ref. [2c]. [d] See
Ref. [2d].
[13] H. Kojima, Y. Urano, K. Kikuchi,
T. Higuchi, Y. Hirata, T. Nagano,
Angew. Chem. 1999, 111, 3419 –
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3422; Angew. Chem. Int. Ed. 1999, 38, 3209 – 3212.
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the DTNB method, except for physostigmine. This is likely a
result of not preincubating ChE and each inhibitor before
commencing the enzymatic reaction in the present method
(all values cited using Ellmanꢀs method were estimated after a
preincubation step). In fact, physostigmine is known to
exhibit strong inhibitory activity against ChE only after
preincubation for rather long periods of time.[19]
In conclusion, almost-nonfluorescent compounds 5 are
transformed effectively to highly fluorescent 3 through
reaction with thiols in aqueous buffer, with significantly
shorter reaction times under mild conditions and without any
side reactions, except for a low level of their hydrolysis. Thus,
compounds 5 may be used as fluorescent probes for thiols and
allow a simple and reliable fluorometric system for measuring
ChE inhibitory activities to be developed. It is believed that
fluorometry with 5 facilitates thiol-quantification assays of
various types of enzymes as a high-throughput technique for
screening enzyme inhibitors as new drugs. Further studies
along these lines are currently underway in our laboratory.
[19] H. Ogura, T. Kosasa, Y. Kuriya, Y. Yamanishi, Methods Find.
Exp. Clin. Pharmacol. 2000, 22, 609 – 613.
Received: January 12, 2005
Published online: April 8, 2005
Keywords: enzymes · fluorescence · high-throughput screening ·
.
inhibitors · sensors
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ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2925