Maolin Guo

Small-molecule-based fluorescent probes, particularly fluorescent analogs of α-amino acids (FAAs), have become valuable tools for labeling proteins of interest and visualizing biological processes in living cells. In this study, we report the design, synthesis, and characterization of a novel cyan-emitting fluorescent α-amino acid, 4-dibenzothiophen-4-yl-L-phenylalanine (DBT-FAA). DBT-FAA was synthesized in good yield via a facile 3-step procedure involving modified Suzuki–Miyaura cross-coupling and characterized by spectroscopic methods. The compound exhibits strong fluorescence in a broad range of aqueous and polar solvents, with a major emission peak at ∼424–445 nm and secondary shoulders at longer wavelengths (∼448–583 nm), along with a high quantum yield (Φ = 0.74 in DMSO at λ_ex = 380 nm and λ_em = 400–800 nm). DBT-FAA also demonstrates excellent photostability, showing strong resistance to photobleaching under aqueous conditions, which is essential for prolonged live-cell imaging. Confocal fluorescence microscopy confirmed that DBT-FAA is efficiently taken up by human HeLa cells, likely through an endocytic pathway, without observable cytotoxicity under the conditions studied. Once inside the cells, DBT-FAA initially accumulates in lysosomes and subsequently translocates to mitochondria with extended incubation time, suggesting dynamic intracellular redistribution. The robust optical properties, photostability, and cellular compatibility of DBT-FAA highlight its potential as a versatile fluorescent amino acid probe for real-time visualization of intracellular processes, offering promising applications in studying protein dynamics, molecular interactions, and cellular metabolism relevant to biochemistry, drug discovery, and protein engineering.

Copper ions are essential trace elements that play critical roles in redox reactions, signal transduction, energy metabolism, and regulation of the central nervous system. However, excess copper can induce cytotoxicity and contribute to various pathological conditions, highlighting the need for sensitive and selective detection methods. We report a novel near-infrared (NIR) optical sensor, IRPhen, based on a heptamethine cyanine scaffold conjugated with a 1,10-phenanthroline Cu²⁺-binding receptor. IRPhen exhibits strong NIR absorption and emission (E_x: 750 nm, E_m: 808 nm), high sensitivity, and good selectivity toward Cu²⁺ over competing metal ions. Spectroscopic studies revealed a rapid, reversible 1:1 binding interaction with a binding constant of 1.3 × 10⁶ M⁻¹ and a detection limit of 0.286 µM. The probe demonstrated excellent stability across physiological pH ranges and maintained its performance under competitive conditions. Importantly, IRPhen is cell-permeable and capable of detecting dynamic Cu²⁺ changes in living fibroblast (WS1) cells using confocal microscopy. This sensor design offers a versatile platform for developing NIR optical sensors to study copper homeostasis, elucidating copper-related biological mechanisms, and potentially developing similar NIR probes for other biologically relevant metal ions.

A convenient tool for detecting iron ions in subcellular structures is desired for better understanding its roles in biological systems. In this work, a new Fe³⁺ sensor, 2-(2-((1-(6-acetylpyridin-2-yl)ethylidene)amino)ethyl)-3',6'-bis(diethylamino)spiro[isoindoline-1,9'-xanthen]-3-one (RhPK), which operates across the entire cellular pH range and is capable of unambiguously detecting Fe³⁺ ion in live human cells at subcellular resolution, is reported. The sensor exhibits high selectivity and sensitivity toward Fe³⁺ with a rapid fluorescence response and a 12-fold increase in intensity upon the addition of 1 equivalent Fe³⁺ at pH 7.3. RhPK forms a 1:1 complex with Fe³⁺ with an apparent binding constant 1.54 × 10⁷ M⁻¹ and a detection limit of 50 nM. The sensor is stable between pH 4.2 and 9.0 and operates across the whole cellular pH range. Cell imaging demonstrates the ability of the sensor to unambiguously detect basal level Fe³⁺ as well as its dynamic changes in real-time in live cells at subcellular resolution, with one labile Fe³⁺ pool identified in mitochondria in human primary fibroblast (ws1) cells for the first time and two Fe³⁺ pools confirmed in mitochondria and endo/lysosomes in human SH-SY5Y neuroblastoma cells, suggesting different cell types have distinctive Fe³⁺ storage in subcellular compartments. The RhPK probe is powerful for rapid and sensitive bioimaging of Fe³⁺ at subcellular level, enabling the unambiguous detection of labile Fe³⁺ pools at the entire cellular pH range, which is of great significance to understand the biological chemistry of Fe³⁺ and its roles in physiological processes and diseases.

We report the facile synthesis and characterization of a novel fluorescent α-amino acid 4-phenanthracen-9-yl-L-phenylalanine (Phen-AA) (5) that emits greenish blue light in the visible region. This genetically encodable L-α-amino acid has excellent photostability with a 75% quantum yield. It readily gets into human cells, being clearly imaged upon 405 nm laser excitation. The synthetic procedure is resistant to racemization and only involves three simple steps which use mild conditions and generate the Phen-AA in reasonably good yield. It may find broad applications in research, biotechnology, and the pharmaceutical industry.

A novel NIR ratiometric fluorescent probe has been developed for monitoring the dynamic changes of subcellular Cu ²⁺ and quantifying its concentration at the nanomolar level in living cells.

A highly selective reaction-based “turn-on” fluorescent sensor is capable of detecting Fe ²⁺ in mitochondria with distinct EPR responses.

Flavonoids have attracted increased attention due to their broad bioactivities related to health and diseases. Modulating metal homeostasis may play an important role in their bioactivities. Recent studies have suggested that dietary flavonoids may affect zinc homeostasis, uptake, and transport. In this work, the zinc-binding sites on a few selected flavonoids have been investigated by ¹H NMR spectroscopy under physiological relevant pH and the species formed were verified by mass spectrometry. Zinc binding induces distinct changes in the proton resonances on the flavonoid rings, providing useful information to locate the Zn-binding sites. No Zn-binding was observed with flavone which lacks a chelation site. Zinc was found to bind to the 3-hydroxyl-4-keto, catechol, and 5-hydroxyl-4-keto chelation sites of flavonol, 3′,4′-dihydroxylflavone and chrysin, respectively. Kaempferol and myricetin chelate zinc at the 3-hydroxyl-4-keto site while rutin binds zinc preferentially at the 5-hydroxyl-4-keto site. However, morin appears to bind zinc at the 1-ether-2-hydroxyl site.

Seeing in many colors: Confocal images acquired using fluorescently labeled amyloid β-peptide revealed its efficient internalization by endocytosis into endosomes/lysosomes of human neuronal cells with a small portion reaching mitochondria, inducing marked cellular and mitochondrial reactive oxygen species production.