Isomerization of (Z,Z) to (E,E) 1-bromo-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene in strong base: Probes for amyloid plaques in the brain

In developing probes for detecting beta -amyloid (A beta) plaques in the br ain of Alzheimer's disease (AD), we have synthesized 1-bromo-2,5-bis-(3-hyd roxycarbonyl-4-hydroxy)styrylbenzene (5, BSB). Due to the presence of two d ouble bonds, formation of four different isomers is possible. Four isomers, E,E-5, E,Z-5, Z,E-5, and Z,Z-5, were prepared. Surprisingly, all showed st rong fluorescent labeling of A beta plaques in the brain of postmortem brai n sections of patients with confirmed AD. In vitro binding assay also showe d that all four isomers of BSB (E,E-5, E,Z-5, Z,E-5, and Z,Z-5) displayed a similar high binding affinity inhibiting the binding of [I-125]E,E, 6, 1-i odo-2,5-bis-(3-hydroxycarbonyl-4-methoxy)styrylbenzene (IMSB) to A beta (1- 40) aggregates. The inhibition constants (K-i) of E,E-5, E,Z-5, E,Z-5, and Z,Z-5 were 0.11 +/- 0.01, 0.19 +/- 0.03, 0.27 +/- 0.06, and 0.13 +/- 0.02 n M, respectively. Due to the fact that geometric stability of these styrylbe nzenes is unknown, and the conversion of Z,Z-5 to E,E-5 may occur automatic ally in the binding or labeling assaying conditions, we have investigated t he kinetics of conversion of Z,Z-5 to E,E-5 by NMR in D2O/NaOD at elevated temperatures (70, 95, and 115 degreesC). The activation energy was determin ed to be 14.15 kcal/mol. The results strongly suggest that the isomeric con version at room temperature in aqueous buffer solution is unlikely. All of the styrylbenzene isomers clearly showed potential as useful tools for stud ying A beta aggregates in the brain. The data suggest that, despite the rig idity of this series of styrylbenzenes, the binding sites on A beta aggrega tes may have certain flexibility and the binding pockets could be adaptable for binding to other smaller ligands. Such information could be exploited to develop new ligands for detecting amyloid plaques in AD.