Brian Corbin

Fluorescent molecular probes for selective and real-time detection of human serum albumin (HSA) protein in urine and blood have important applications in clinical diagnostics for various diseases. The majority of known fluorescent HSA probes give emission in the blue-green spectral region where biological autofluorescence could interfere with the detection. In this study, a series of chalcones with extended conjugation are shown to give red-emission with a large Stokes shift (λ_abs ≈ 430–445 nm, λ_em ≈ 620–640 nm), and low fluorescence in H₂O. Upon addition of HSA protein, the fluorescence of these chalcones is shown to increase significantly, by as high as 30-fold. Potential site-selective binding to HSA has been examined by competitive displacement experiments using various known site-selective drugs. In conjunction with molecular docking experiments, the study further indicates that the chalcones could selectively enter the FA1 (Fatty Acid site 1) binding pocket (Drug Site III).

4-Dimethylamino-2′-hydroxy chalcone (DHC) 1 is an important natural compound that is nearly non-fluorescent in solution but highly fluorescent in its crystalline state. At room temperature, the weak fluorescence from the DHC solution is exclusively from its keto tautomer, without notable contribution from its enol tautomer. By using low-temperature fluorescence, the study found that the enol emission could be detected upon cooling with liquid N₂ in a protic solvent (e.g., EtOH). This led to observation of the fluorescence vibronic structure of enol tautomer, in addition to its enol emission λ_em ≈ 473 nm that is well separated from its keto tautomer emission (λ_em ≈ 600 nm). By freezing DHC in a solvent matrix, the study revealed the fluorescent characteristics of a single molecule in a rigid environment. Further comparison of DHC in a solvent matrix and crystalline state disclosed that the emission of crystalline DHC was primarily from the keto tautomer, along with some minor contribution from the enol tautomer, despite the tight packing environment in the crystalline state.

4-Dimethylamino-2’-hydroxy chalcone (DHC) 1 is an important natural compound that is nearly non-fluorescent in solution but highly fluorescent in its crystalline state. At room temperature, the weak fluorescence from DHC solution is exclusively from its keto tautomer, without notable contribution from its enol tautomer. By using low temperature fluorescence, the study found that the enol emission could be detected upon cooling with liquid N₂ in a protic solvent (e.g. EtOH). This led to observation of the fluorescence vibronic structure of enol tautomer, in addition to its enol emission λ_em ≈ 473 nm that is well separated from its keto tautomer emission (λ_em ≈ 600 nm). By freezing DHC in a solvent matrix, the study revealed the fluorescent characteristics of a single molecule in a rigid environment. Further comparison of DHC in a solvent matrix and crystalline state disclosed that the emission of crystalline DHC was primarily from the keto tautomer, along with some minor contribution from the enol tautomer, despite the tight packing environment in the crystalline state.

The chalcone derivatives with hydroxy group (2) have been examined using low-temperature fluorescence spectroscopy. The study aimed to freeze the intramolecular charge transfer (ICT) motion in order to reveal the potential hidden transition(s) that are difficult to observe at room temperature. Although chalcone 2 revealed one emission peak at ~667 nm at room temperature, it exhibited two emission peaks (λ_em = 580 and 636 nm) in EtOH at liquid N₂ temperatures (77 K). With the aid of model compound 3 with methoxy group and aluminum complex 2-Al³⁺, attempts were made to assign these emission peaks. The results point towards the possibility of the coexistence of ICT and excited state intramolecular proton transfer (ESIPT) in the chalcone derivatives with extended conjugation.

Two NIR-emitting donor-π-acceptor (D-π-A) type regioisomeric styryl pyridinium dyes (1a–1b) were synthesized and studied for their photophysical performance and environment sensitivity. The two regioisomers, 1a and 1b, exhibited interesting photophysical properties including, longer wavelength excitation (λ_ex ≈ 530–560 nm), bright near-infrared emission (λ_em ≈ 690–720 nm), high-fluorescence quantum yields (ϕ_fl ≈ 0.24–0.72) large Stokes’ shift (∆λ ≈ 150–240 nm) and high-environmental sensitivity. Probe’s photophysical properties were studied in different environmental conditions such as polarity, viscosity, temperature, and concentration. Probes (1a–1b) exhibited noticeable changes in absorbance, emission and Stokes’ shift while responding to the changes in physical environment. Probe 1b exhibited a significant bathochromic shift in optical spectra (∆λ ≈ 20–40 nm) compared to its isomer 1a, due to the regio-effect. Probes (1a–1b) exhibited an excellent ability to visualize bacteria (Bacillus megaterium, Escherichia coli), and yeast (Saccharomyces cerevisiae) via fluorescence microscopy.

Accurate determination of Cu²⁺ in solution is crucial for preventing several disease conditions. Spectroscopy-based techniques for metal ion detection are promising methods due to their excellent sensitivity and rapid response time. In this work, we are reporting a newly synthesized 2-(2′-Hydroxyphenyl) benzoxazole-based compound, probe 2, by incorporating a vinyl pyridinium segment into the bis(HBO) 4 system. Probe 2 exhibited excellent specificity toward Cu²⁺ in solution. The ratiometric absorbance (λ₄₄₀/λ₃₇₀) and the quenching of fluorescence at λ_em ≈585 nm exhibited an excellent linear correlation. The formation of the 2-Cu complex can be utilized as a highly sensitive spectroscopic method for the detection of Cu²⁺ in solution with a detection limit of 0.15 µM. In addition, Cu²⁺-induced fluorescence quenching in probe 2 occurs mainly via a static quenching mechanism by forming a 2-Cu complex, and the stability constant for the 2-Cu complex was calculated based on spectroscopic measurements.

Two interesting benzothizolium-based D-π-A type hemicyanine dyes (3a–3b) with a diphenylamine (-NPh₂) donor group were evaluated for fluorescence confocal microscopy imaging ability in live cells (MO3.13, NHLF). In sharp contrast to previously reported D-π-A dyes with alkyl amine donor (-NR₂) groups (1), 3a and 3b exhibited significantly different photophysical properties and organelle selectivity. Probes 3a and 3b were nearly non-fluorescent in many polar and non-polar solvents but exhibited a bright red fluorescence (λ_em ≈ 630–640 nm) in stained MO3.13 and NHLF with very low probe concentrations (i.e., 200 nM). Fluorescence confocal microscopy-based co-localization studies revealed excellent lysosome selectivity from the probes 3a–3b, which is in sharp contrast to previously reported D-π-A type benzothiazolium dyes (1) with an alkyl amine donor group (-NR₂) (exhibiting selectivity towards cellular mitochondria). The photostability of probe 3 was found to be dependent on the substituent (R’) attached to the quaternary nitrogen atom in the cyanine dye structure. The observed donor-dependent selectivity switching phenomenon can be highly useful in designing novel organelle-targeted fluorescent probes for live-cell imaging applications.