In this study inspired by previous work on 3-substituted Asp analogues, we designed and synthesized a total of 32 β-sulfonamide Asp analogues and characterized their pharmacological properties at the excitatory amino acid transporter subtypes EAAT1, EAAT2, and EAAT3. In addition to several potent EAAT inhibitors displaying IC values ∼1 μM at all three subtypes, this elaborate structure-activity relationship also identified analogues exhibiting distinct preferences or selectivities for specific transporter subtypes. Introduction of two fluorine atoms on the phenyl ring yielded analogue 4y that displayed an IC of 0.8 μM at EAAT1 with a 14- and 9-fold preference over EAAT2 and EAAT3, respectively. Conversely, the m-CF-phenyl analogue 4r was a potent selective EAAT2-inhibitor (IC = 2.8 μM) exhibiting 30- and 50-fold selectivity over EAAT1 and EAAT3, respectively. In conclusion, even small structural differences in these β-sulfonamide Asp analogues provide analogues with diverse EAAT subtype selectivity profiles.

Increasing demand for detecting single molecules in challenging environments has raised the bar for the fluorophores used. To achieve better resolution and/or contrast in fluorescence microscopy, it is now essential to use bright and stable dyes with tailored photophysical properties. While long fluorescence lifetime fluorophores offer many advantages in time-resolved imaging, their inherently lower molar absorption coefficient has limited applications in single molecule imaging. Here we propose a generic approach to prepare bright, long fluorescence lifetime dyad fluorophores comprising an absorbing antenna chromophore with high absorption coefficient linked to an acceptor emitter with a long fluorescence lifetime. We introduce a dyad consisting of a perylene antenna and a triangulenium emitter with 100% energy transfer from donor to acceptor. The dyad retained the long fluorescence lifetime (∼17 ns) and high quantum yield (75%) of the triangulenium emitter, while the perylene antenna increased the molar absorption coefficient (up to 5 times) in comparison to the free triangulenium dye. These triangulenium based dyads with significantly improved brightness can now be detected at the single molecule level and easily discriminated from bright autofluorescence by time-gated and other lifetime-based detection schemes.

Four‐stranded G‐quadruplex (G4) DNA is a non‐canonical DNA topology that has been proposed to form in cells and play key roles in how the genome is read and used by the cellular machinery. Previously, a fluorescent triangulenium probe (DAOTA‐M2) was used to visualise G4s in cellulo, thanks to its distinct fluorescence lifetimes when bound to different DNA topologies. Herein, the library of available triangulenium probes is expanded to explore how modifications to the fluorescent core of the molecule affect its photophysical characteristics, interaction with DNA and cellular localisation. The benzo‐bridged and isopropyl‐bridged diazatriangulenium dyes, BDATA‐M2 and CDATA‐M2 respectively, featuring ethyl‐morpholino substituents, were synthesised and characterised. The interactions of these molecules with different DNA topologies were studied to determine their binding affinity, fluorescence enhancement and fluorescence lifetime response. Finally, the cellular uptake and localisation of these optical probes were investigated. Whilst structural modifications to the triangulenium core only slightly alter the binding affinity to DNA, BDATA‐M2 and CDATA‐M2 cannot distinguish between DNA topologies through their fluorescence lifetime. It is argued theoretically and experimentally that this is due to reduced effectiveness of photoinduced electron transfer (PET) quenching. This work presents valuable new evidence into the critical role of PET quenching when using the fluorescence lifetime of triangulenium dyes to discriminate G4 DNA from duplex DNA, highlighting the importance of fine tuning redox and spectral properties when developing new triangulenium‐based G4 probes.

The lipophilic natureo fo rganic dyes complicates their effectiveness in aqueous solutions. In this work we investigate threed ifferent strategies for achieving water-solubility of the diazaoxatriangulenium (DAOTA +)c hromophore: hydrophilic counter ions, aromatic sulfonationo ft he chromophore, and attachment of charged side chains. The long fluorescence lifetime(FLT, t f = 20 ns) of DAOTA + makes it a sensitive probet oa nalyze solvation and aggregation effects. Direct sulfonation of the chromophore was found to increase solubility drastically,b ut at the cost of greatly re-ducedq uantum yields (QYs) due to enhanced non-radiative deactivation processes. The introduction of either cationic (4) or zwitterionic side chains (5), however,b rings the FLT (t f = 18 ns) and QY (f f = 0.56) of the dye to the same level as the parent chromophorei na cetonitrile. Time-resolved fluorescence spectroscopya lso reveals ah igh resistance to aggregation and non-specific binding in ah igh loading of bovines erum albumin (BSA). The results clearly show that addition of charged flexibles ide chains is preferable to directs ulfonation of the chromophore core.

Access to functionalization of new sites on the triangulenium core structure has been achieved at an early stage by chlorination with N-chlorosuccinimide (NCS), giving rise to two new triangulenium dyes (1 and 2). By introducing the chlorine functionalities in the acridinium precursor, positions complementary to those previously accessed by electrophilic aromatic substitution of the final dyes can be accesed. The chlorination is selective, giving only one regioisomer for both mono-and dichlorination products. For the monochlorinated acridinium compound a highly selective ring-closing reaction was discovered to generate only a single regioisomer of the cationic [4]helicene product. This discovery aspired further investigations into the mechanism of [4]helicene formation and to the first isolation of the previously proposed intermediate of the two-step SNAr reaction, key to all aza-bridged triangulenium and helicenium systems. A late stage functionalization of DAOTA + with NCS gave rise to a different dichlorinated compound (2). The fully ring closed chlorinated triangulenium dyes 1, 2 and 3 show a redshift in absorption and emission relative to the non-chlorinated analogues, while still maintaining relatively high fluorescence quantum yields of 36%, 26%, and 41%, and long fluorescence lifetimes of 15 ns, 12.5 ns and 16 ns, respectively. Cyclic voltammetry shows that chlorination of the triangulenium dyes significantly lowers reduction potentials and thus allows for efficient tuning of redox and photo-redox properties.