Protein deposits are associated with many devastating diseases and fluorescent ligands able to visualize these pathological entities are essential. Here, we report the synthesis of thiophene‐based donor–acceptor–donor heptameric ligands that can be utilized for spectral assignment of distinct amyloid‐β (Aβ) aggregates, one of the pathological hallmarks in Alzheimer's disease. The ability of the ligands to selectively distinguish Aβ deposits was abolished when the chemical composition of the ligands was altered. Our findings provide the structural and functional basis for the development of new fluorescent ligands that can distinguish between aggregated proteinaceous species consisting of the same peptide or protein. In addition, such ligands might aid in interpreting the potential role of polymorphic Aβ deposits in the pathogenesis of Alzheimer's disease.

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.

Optotracing is a novel method for analytical imaging of carbohydrates in plant and microbial tissues. This optical method applies structure-responsive oligothiophenes as molecular fluorophores emitting unique optical signatures when bound to polysaccharides. Herein, we apply Carbotrace TM 680, a short length anionic oligothiophene with a central heterocyclic benzodithiazole (BTD) motif, to probe for different glucans. The donor-acceptor-donor type electronic structure of Carbotrace TM 680 provides improved spectral properties compared to oligothiophenes due to the possibility of intramolecular chargetransfer transition to the BTD motif. This enables differentiation of glucans based on the glycosidic linkage stereochemistry. Thus a-configured starch is readily differentiated from b-configured cellulose. The versatility of optotracing is demonstrated by dynamic monitoring of thermo-induced starch remodelling, shown in parallel by spectrophotometry and microscopy of starch granules. Imaging of Carbotrace TM 680 bound to multiple glucans in plant tissues provided direct identification of their physical locations, revealing the spatial relationship between structural (cellulose) and storage (starch) glucans at sub-cellular scale. Our work forms the basis for the development of superior optotracers for sensitive detection of polysaccharides. Our non-destructive method for anatomical mapping of glucans in biomass will serve as an enabling technology for developments towards efficient use of plant-derived materials and biomass.

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 areaunder-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 onswitch 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.

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 Alzheimer's disease (AD). The ability of the ligands to selectively identify AD‐associated pathological alterations, such as deposits composed of aggregates of the amyloid‐β (Aβ) 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.