Synthesis and Screening of a Library of Re/Tc-Based Amyloid Probes Derived from -Breaker Peptides Karin A. Stephenson, † Leslie C. Reid, † Jon Zubieta, ‡ John W. Babich, § Mei-Ping Kung, ⊥ Hank F. Kung, ⊥ and John F. Valliant* ,† Departments of Chemistry and Medical Physics & Applied Radiation Sciences, McMaster University, Hamilton, ON, Canada, L8S 4M1, Department of Chemistry, Syracuse University, Syracuse, New York 13244-4100, Molecular Insight Pharmaceuticals Inc., Cambridge, Massachusetts 02142, and Department of Radiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104. Received January 2, 2008; Revised Manuscript Received February 13, 2008 Through the development and application of a unique approach for producing Re-metallopeptides, a new class of peptide-derived probes that are designed to target -amyloid plaques was developed. Derivatives of a class of -breaker peptides having the core sequence lvffa or affvl (lower case letters represent D-amino acids) and the single amino acid chelate quinoline (SAACQ) ligand which can bind Re and 99m Tc were prepared on an automated peptide synthesizer. Both monomeric and dimeric peptides were synthesized in modest to good yields where in select examples a biotin-containing amino acid derivative was included to act as a linker point for further conjugation to carrier proteins. The Re complexes for all reported peptides were prepared similarly and screened for their ability to inhibit fibrillogenesis. Two of the reported compounds showed excellent inhibitory properties (8a: 40 ( 5% amyloid formation versus control; 16: 40 ( 4%) and warrant further investigation. For one of these leads, the 99m Tc analogue was synthesized and the product showed high stability toward histidine and cysteine challenges, making it a viable candidate for in ViVo biodistribution studies. INTRODUCTION Alzheimer’s disease (AD) is a progressive neurodegenera- tive affliction that involves the formation of plaques and neurofibrillary tangles, which ultimately lead to neuronal death in both the hippocampus and frontal cortex (1, 2). Senile plaques can occupy up to 15% of the brain volume in some advanced AD patients and are composed primarily of -amyloid peptides (A 1-40,42,43 )(1, 3). Amyloid peptides are naturally occurring entities, but in AD patients, the peptides form plaques in the brain: a process that stems from a change in the mechanism of cleavage of the amyloid precursor protein (1, 4, 5). The formation of senile plaques has been correlated with the progression of the disease and has therefore emerged as a promising target for the treatment of AD (1, 6, 7). There has been a major effort put into developing radioim- aging agents that allow for serial monitoring of the progression of AD and for assessing the impact of therapy. 18 F-deoxyglucose (FDG) is used to diagnose the early onset of disease by visualizing reduced metabolism of glucose associated with cell death in the brains of AD patients (2). Unfortunately, FDG is not able to specifically identify -amyloid plaques; therefore, to better characterize changes in plaque load, work in the field has been geared toward developing plaque-targeting PET and SPECT agents (8-12). Maggio and co-workers reported that A 1-40 labeled with 125 I rapidly deposits on -amyloid plaques in isolated tissue. Not surprisingly, the labeled peptide showed particularly poor brain uptake in ViVo (13). However, conjugation of an A 1-40 - biotin derivative to a streptavidin-tagged monoclonal antibody (mAb) for the human insulin receptor markedly improved transport across the BBB (14, 15). Uptake of the mAb- 125 I- biotin-A 1-40 conjugate in rhesus monkey brains was observed using autoradiography. Structure-activity relationships (SARs) have shown that fragments of the full A 1-40 peptide can exhibit binding to plaques and inhibition of -amyloid fibrillogenesis (4, 16-21). -sheet breaker peptides having as few as four amino acids (LVFF) have been able to significantly inhibit fibril formation (22). The key hydrophobic motif, LVFF, which is common to most AD -sheet breaker peptides, binds amyloid plaques and can prevent soluble A peptides from forming structured aggregates (4, 16-19). Given that strategies for enabling peptides to cross the BBB exist, radiotracers based on -breaker peptides would offer a new means for imaging -amyloid plaques and for assessing therapies aimed at solubilizing plaques. The traditional approach for developing 99m Tc-labeled probes of this type would involve appending a technetium chelator to the N-terminus of a core -breaker peptide sequence. After isolation, attempts would be made to prepare the rhenium complex of the product, which would serve as a reference standard for the 99m Tc analogue and as a compound to test the ability of the metallopeptide derivative to bind plaques and/or inhibit amyloid fibrillogenesis. This linear discovery approach is time-consuming with respect to isolating and fully character- izing the Re complexes and identifying the optimal sequence for the vector and site of derivatization on the peptide backbone. We report here a new approach in which an organometallic complex of an amino acid analogue is used to insert the metal into various positions in lvffa and affvl sequences. A synthetic methodology was developed such that multivalent (i.e., dimeric) peptides could be prepared and biotin incorporated into the backbone of the peptide as a linker site for conjugation to carrier proteins. The ability of the Re complexes to prevent fibril * To whom correspondence should be addressed. Tel: 905-525-9140, ext. 22840. FAX: 905-522-2509. E-mail: valliant@mcmaster.ca. † McMaster University. ‡ Syracuse University. § Molecular Insight Pharmaceuticals Inc. ⊥ University of Pennsylvania. Bioconjugate Chem. 2008, 19, 1087–1094 1087 10.1021/bc800001g CCC: $40.75  2008 American Chemical Society Published on Web 04/12/2008