No abstract

Senile plaques (SPs) and neurofibrillary tangles (NFTs) are hallmark pathologies accompanying the neurodegeneration involved in Alzheimer's disease (AD), for which beta-amyloid (Abeta) peptide is a major constituent of SPs. Our laboratories previously developed the hydrophobic, fluorescent molecular-imaging probe 2-(1-(6-[(2-[(18)F]fluoroethyl)(methyl)amino]-2-naphthyl)ethylidene)malononitrile ([(18)F]FDDNP), which crosses the blood-brain barrier and determines the localization and load of SPs and NFTs in vivo in AD patients. In this report, we used fluorimetric and radioactive binding assays to determine the binding affinities of FDDNP and its analog, 1-(6-[(2-[(18)F]fluoroethyl)(methyl)amino]naphthalen-2-yl)ethanone ([(18)F]FENE), to synthetic fibrils of Abeta(1-40). FDDNP and FENE both appeared to bind to two kinetically distinguishable binding sites on Abeta(1-40) fibrils. Fluorescence titrations yielded apparent K(d) values of 0.12 and 0.16 nm for high-affinity binding sites for FDDNP and FENE, respectively, and apparent K(d) values of 1.86 and 71.2 nm for the low-affinity binding sites. The traditional radioactive binding assays also produced apparent K(d) values in the low nanomolar range. The presence of two kinetically distinguishable binding sites for FDDNP and FENE suggests multiple binding sites for SPs and identifies the parameters that allow for the structural optimization of this family of probes for in vivo use. The high-affinity binding of the probes to multiple binding sites on fibrils are consistent with results obtained with digital autoradiography, immunohistochemistry, and confocal fluorescence microscopy using human brain specimens of AD patients.

1,1-Dicyano-2-[6-(dimethylamino)naphthalen-2-yl]propene (II, DDNP) has been synthesized as a fluorescent dye whose intramolecular rotational relaxation is solvent polarity and viscosity dependent. Its fluorescence emission (λ max: 470-610 nm in hydrophobic and viscous environments) is in a very favorable region for its use with visible fluorescence microscopy, without interference from cell or tissue autofluorescence. Its fluorescence emission quantum yield in an aqueous environment is very low, allowing for facile differentiation between lipid or protein bound state and aqueous media. II crystallizes into two solid isoforms containing one (red crystals) and two conformers (yellow crystals), exhibiting differing spectral properties. X-ray crystal structures reveal a planar arrangement between dimethylamino and naphthalenyl moieties, with out-of-plane arrangements between the malononitrile and naphthalene ring found in each of the three conformers.

The biomarker 2-(1-{6-[(2-[(18)F]fluoroethyl)(methyl)amino]-2-naphthyl}ethylidene)malononitrile ([(18)F]FDDNP) is used as a positron emission tomography (PET) imaging probe for Alzheimer's disease and other neurodegenerative diseases. A high-yield and fully automated synthesis of [(18)F]FDDNP--along with the synthesis and characterization of non-radioactive FDDNP, a fluorescent probe derived from 2-(1,1-dicyanopropenyl-2)-6-dimethylaminonaphthalene (DDNP)--are reported. Radiofluorination of the tosyloxy precursor 2-{[6-(2,2-dicyano-1-methylvinyl)-2-naphthyl](methyl)amino}ethyl-4-methylbenzenesulfonate (DDNPTs) with K(18)F/Kryptofix 2.2.2. yielded chemically (>99%) and radiochemically (>99%) pure [(18)F]FDDNP in high radiochemical yields (40-60%; n> 120), with specific activities ranging from 4 to 8 Ci/mumol at the end of synthesis (90 minutes). Both remote, semiautomated and automated synthesis procedures are described. Either approach provides a reliable method for production of large quantities (110-170 mCi from 500 mCi of [(18)F]fluoride) of [(18)F]FDDNP allowing for multiple PET experiments in the same day or for distribution of the tracer from a single cyclotron facility to PET imaging centers at various geographical distances.

The positron-emission tomography (PET) probe 2-(1-[6-[(2-fluoroethyl)(methyl)amino]-2-naphthyl]ethylidene) (FDDNP) is used for the noninvasive brain imaging of amyloid-β (Aβ) and other amyloid aggregates present in Alzheimer’s disease and other neurodegenerative diseases. A series of FDDNP analogs has been synthesized and characterized using spectroscopic and computational methods. The binding affinities of these molecules have been measured experimentally and explained through the use of a computational model. The analogs were created by systematically modifying the donor and the acceptor sides of FDDNP to learn the structural requirements for optimal binding to Aβ aggregates. FDDNP and its analogs are neutral, environmentally sensitive, fluorescent molecules with high dipole moments, as evidenced by their spectroscopic properties and dipole moment calculations. The preferred solution-state conformation of these compounds is directly related to the binding affinities. The extreme cases were a nonplanar analog t -butyl-FDDNP, which shows low binding affinity for Aβ aggregates (520 nM K i ) in vitro and a nearly planar tricyclic analog cDDNP, which displayed the highest binding affinity (10 pM K i ). Using a previously published X-ray crystallographic model of 1,1-dicyano-2-[6-(dimethylamino)naphthalen-2-yl]propene (DDNP) bound to an amyloidogenic Aβ peptide model, we show that the binding affinity is inversely related to the distortion energy necessary to avoid steric clashes along the internal surface of the binding channel.