Aggregated species of amyloid-beta (A beta) are one of the pathological hallmarks in Alzheimer's disease (AD), and ligands that selectively target different A beta deposits are of great interest. In this study, fluorescent thiophene-based ligands have been used to illustrate the features of different types of A beta deposits found in AD brain tissue. A dual-staining protocol based on two ligands, HS-276 and LL-1, with different photophysical and binding properties, was developed and applied on brain tissue sections from patients affected by sporadic AD or familial AD associated with the PSEN1 A431E mutation. When binding to A beta deposits, the ligands could easily be distinguished for their different fluorescence, and distinct staining patterns were revealed for these two types of AD. In sporadic AD, HS-276 consistently labeled all immunopositive A beta plaques, whereas LL-1 mainly stained cored and neuritic A beta deposits. In the PSEN1 A431E cases, each ligand was binding to specific types of A beta plaques. The ligand-labeled A beta deposits were localized in distinct cortical layers, and a laminar staining pattern could be seen. Biochemical characterization of the A beta aggregates in the individual layers also showed that the variation of ligand binding properties was associated with certain A beta peptide signatures. For the PSEN1 A431E cases, it was concluded that LL-1 was binding to cotton wool plaques, whereas HS-276 mainly stained diffuse A beta deposits. Overall, our findings showed that a combination of ligands was essential to identify distinct aggregated A beta species associated with different forms of AD.

The aggregation and accumulation of proteins in the brain is the defining feature of many devastating neurodegenerative diseases. The development of fluorescent ligands that bind to these accumulations, or deposits, is essential for the characterization of these neuropathological lesions. We report the synthesis of donor-acceptor-donor (D-A-D) thiophene-based ligands with different emission properties. The D-A-D ligands displayed selectivity towards distinct disease-associated protein deposits in histological sections from postmortem brain tissue of individuals affected by Alzheimers disease (AD). The ability of the ligands to selectively identify AD-associated pathological alterations, such as deposits composed of aggregates of the amyloid-beta (A beta) peptide or tau, was reduced when the chemical composition of the ligands was altered. When combining the D-A-D ligands with conventional thiophene-based ligands, superior spectral separation of distinct protein aggregates in AD tissue sections was obtained. Our findings provide the structural and functional basis for the development of new fluorescent ligands that can distinguish between aggregated proteinaceous species, as well as offer novel strategies for developing multiplex fluorescence detection of protein aggregates in tissue sections.

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 Alzheimers disease (AD) pathology. The selectivity of the ligands towards AD associated pathological hallmarks, such as aggregates of the amyloid-beta (A beta) 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 thiophene-based ligands that can be utilized for optical assignment of different aggregated proteinaceous species in tissue sections.

Optotracers are conformation-sensitive fluorescent tracer molecules that detect peptide- and carbohydrate-based biopolymers. Their binding to bacterial cell walls allows selective detection and visualisation of Staphylococcus aureus (S. aureus). Here, we investigated the structural properties providing optimal detection of S. aureus. We quantified spectral shifts and fluorescence intensity in mixes of bacteria and optotracers, using automatic peak analysis, cross-correlation, and area-under-curve analysis. We found that the length of the conjugated backbone and the number of charged groups, but not their distribution, are important factors for selective detection of S. aureus. The photophysical properties of optotracers were greatly improved by incorporating a donor-acceptor-donor (D-A-D)-type motif in the conjugated backbone. With significantly reduced background and binding-induced on-switch of fluorescence, these optotracers enabled real-time recordings of S. aureus growth. Collectively, this demonstrates that chemical structure and photophysics are key tunable characteristics in the development of optotracers for selective detection of bacterial species.

Thiophene based ligands represent a class of molecular reporters proven superior in discerning pathological targets involved in neurodegenerative diseases, as well as bacterial infection. By fluorometric detection that depends on the milieu surrounding the ligand these biological processes can be studied with fluorescence spectroscopy and hyper-spectral confocal microscopy. The binding of a thiophene-based ligand to a biological target can entail specific fluorescent read-out through the conformation-sensitive ligand. Thus, the photo-physical properties of these molecules and the optical connection of binding make them valuable tools in the study of pathological events. As optical detection of pathological phenomena can be realized through several fluorescence parameters, including changes in fluorescent intensity, wavelength shifts, energy transfer, or emission lifetime, molecular studies of pathological targets in several biological systems have been realized by employing thiophene-based ligands. For instance, utilization of conjugated polydisperse and monodisperse thiophene-based molecules has in several studies demonstrated detection of disease associated protein aggregates in vitro, ex vivo and in vivo. 

My doctoral studies have included the synthesis and characterization of thiophene based donor-acceptor-donor (D–A–D) molecules and evaluation of how changes in side-chain positions, molecular length and number of negatively charged carboxylates impact interaction with biological targets. This thesis describes the utilization of the D–A–D molecules as fluorescent ligands for protein aggregates associated with Alzheimer’s disease and optical assignment of specific bacteria. The design and synthesis of these novel D–A–D thiophene-based fluorophores, with alterations in back bone, distribution of side chains and negatively charged groups, have generated novel insights regarding the ligands chemical structure on ligand performance, by the assessment of binding mode of the respective ligand to distinct pathological entities. Furthermore, the D–A–D molecules hold alternative photo-physical properties compared to thiophene-based ligand and these optical properties of the ligands have been employed to provide new insights in questions regarding protein aggregate polymorphism in Alzheimer’s disease. Overall, by organic synthesis we have fine-tuned the properties of thiophene-based D-A-D molecules and evaluated how modifications affect interactions with distinct biological, pathological targets and we foresee that D–A–D thiophene-based ligands will expand the toolbox for studying pathological targets in neurodegenerative diseases, as well as bacterial infection. 

By attaching an azide functional group via a tetraethylene glycol linker to the α-terminal position of a variety of oligothiophenes, thiophene-based ligands that can be utilized for detection of protein aggregates with surface plasmon resonance have been developed. All ligands displayed selectivity towards recombinant amyloid fibrils and the LCO/protein aggregate interaction could be detected by fluorescence as well as by surface plasmon resonance.