Proteophenes – Amino Acid Functionalized Thiophene‐based Fluorescent Ligands for Visualization of Protein Deposits in Tissue Sections with Alzheimer's Disease Pathology

Abstract

Protein deposits composed of specific proteins or peptides are associated with several neurodegenerative diseases and fluorescent ligands able to detect these pathological hallmarks are vital. Here, we report the synthesis of a class of thiophene-based ligands, denoted proteophenes, with different amino acid side-chain functionalities along the conjugated backbone, which display selectivity towards specific disease-associated protein aggregates in tissue sections with Alzheimer's disease (AD) pathology. The selectivity of the ligands towards AD associated pathological hallmarks, such as aggregates of the amyloid-β (Aβ) peptide or tau filamentous inclusions, was highly dependent on the chemical nature of the amino acid functionality, as well as on the location of the functionality along the pentameric thiophene backbone. Finally, the concept of synthesizing donor-acceptor-donor proteophenes with distinct photophysical properties was shown. Our findings provide the structural and functional basis for the development of new thiophenebased ligands that can be utilized for optical assignment of different aggregated proteinaceous species in tissue sections.

Thiophene‐Based Ligands: Design, Synthesis and Their Utilization for Optical Assignment of Polymorphic‐Disease‐Associated Protein Aggregates

“…[122,175,176] In addition, due to minor chemical variations of the ligands, chemical determinants for achieving superior ligands for optical separation of distinct protein aggregates have been identified. [115,117,118,120,121,125,127] In 2015, Hermann and co-workers showed that p-FTAA (Figure 4A) binds to amyloid fibrils in a similar way as the conventional ligand CR. [122] By using the prion-forming domain of the fungal HET-s prion as a model, solid-state nuclear magnetic resonance analyses and molecular dynamics simulations showed that anionic side chains of p-FTAA interacted with complementary, regularly spaced lysine residues situated in well-accessible grooves along the HET-s fibrils (Figure 12A).…”

“…Such a dual staining protocol, allowing multiplex photophysical detection of distinct protein aggregates in brain tissue sections with AD pathology, was afforded when combining two proteophenes, HS-84-V-E (Figure 3E) and HS-169-V-V, with distinct amino acid side-chain functionalities along the conjugated thiophene backbone. [127] As the selectivity of the proteophenes towards AD associated pathological hallmarks was highly dependent on the chemical nature, as well as on the location of the amino acid functionality, a differential staining of Aβ or tau aggregates was observed. Characteristic lifetime distributions (600 and 1000 ps) from HS-84-V-E were observed from Aβ cored plaques, diffuse Aβ plaques, cerebral amyloid angiopathy (CAA) lesions and tau pathology, whereas longer decay times, 2000-3000 ps, associated with HS-169-V-V could only be detected from CAA lesions (Figure 7B).…”

“…In addition, the concept of synthesizing D-A-D proteophenes with distinct photophysical properties was also shown. [127] Overall, the chemical evolution of thiophene-based ligands from polydisperse LCPs to a diversity of chemically defined LCOs (Figures 3 and 4) has enhanced the toolbox of fluorescent ligands for optical assignment of disease-associated protein aggregates and in the next section, we will illustrate how both LCPs and LCOs have been applied to study these pathological entities in a variety of disease models as well as in patient derived tissue sections.…”

“…Lately, the LCO-FLIM methodology has been evolved further by using combinations of LCOs and D-A-D-based LCOs. [127,147] As D-A-D ligands display completely different fluorescence decay times compared to pure thiophene-based LCOs, [126] combinations of ligands can be For PTAA, spectra with emission maxima at 570 (blue), 580 (green), or 590 nm (red) were observed. The wavelengths used for calculation of the spectral ratios R 538=Emax and R 538/642 , 538 nm (*), E max (+) and 642 nm (À ), respectively, are highlighted.…”

“…[125] Further correlations between ligand binding mode to distinct protein aggregates and chemical composition of the ligand were afforded when using proteophenes for staining of human AD brain tissue sections. [127] As mentioned above, proteophenes are the latest generation of LCOs with specific amino acid side-chain functionalization along the thiophene backbone. In addition to the α-carbon side chain from the amino acids, the carboxyl groups of the amino acids are also accessible since the amino acid was linked to the thiophene moieties with HATU-mediated amide coupling reactions.…”