Synthesis of Firefly Luciferin Analogues and Evaluation of the Luminescent Properties

Five new firefly luciferin ( 1 ) analogues were synthesized and their light emission properties were examined. Modifications of the thiazoline moiety in 1 were employed to produce analogues containing acyclic amino acid side chains ( 2 – 4 ) and heterocyclic rings derived from amino acids ( 5 and 6 ) linked to the benzothiazole moiety. Although methyl esters of all of the synthetic derivatives exhibited chemiluminescence activity, only carboluciferin ( 6 ), possessing a pyrroline‐substituted benzothiazole structure, had bioluminescence (BL) activity (λ_max=547 nm). Results of bioluminescence studies with AMP‐carboluciferin (AMP=adenosine monophosphate) and AMP‐firefly luciferin showed that the nature of the thiazoline mimicking moiety affected the adenylation step of the luciferin–luciferase reaction required for production of potent BL. In addition, BL of 6 in living mice differed from that of 1 in that its luminescence decay rate was slower.     

Rare‐Earth‐Based Nanoparticles with Simultaneously Enhanced Near‐Infrared (NIR)‐Visible (Vis) and NIR‐NIR Dual‐Conversion Luminescence for Multimodal Imaging

Multifunctional NaGdF₄:Yb³⁺,Er³⁺,Nd³⁺@NaGdF₄:Nd³⁺ core–shell nanoparticles (called Gd:Yb³⁺,Er³⁺,Nd³⁺@Gd:Nd³⁺ NPs) with simultaneously enhanced near‐infrared (NIR)‐visible (Vis) and NIR‐NIR dual‐conversion (up and down) luminescence (UCL/DCL) properties were successfully synthesized. The resulting core–shell NPs simultaneously emitted enhanced UCL at 522, 540, and 660 nm and DCL at 980 and 1060 nm under the excitation of a 793 nm laser. The enhanced UCL and DCL can be explained by complex energy‐transfer processes, Nd³⁺→Yb³⁺→Er³⁺ and Nd³⁺→Yb³⁺, respectively. The effects of Nd³⁺ concentration and shell thickness on the UCL/DCL properties were systematically investigated. The UCL and DCL properties of NPs were observed under the optimal conditions: a shell Nd³⁺ content of 20 % and a shell thickness of approximately 5 nm. Moreover, the Gd:Yb³⁺,Er³⁺,Nd³⁺@Gd:20 % Nd³⁺ NPs exhibited remarkable magnetic resonance imaging (MRI) properties similar to that of a clinical agent, Omniscan. Thus, the core–shell NPs with excellent UCL/DCL/magnetic resonance imaging (MRI) properties have great potential for both in vitro and in vivo multimodal bioimaging.     

Firefly Luciferase Mutants Allow Substrate‐Selective Bioluminescence Imaging in the Mouse Brain

Bioluminescence imaging is a powerful approach for visualizing specific events occurring inside live mice. Animals can be made to glow in response to the expression of a gene, the activity of an enzyme, or the growth of a tumor. But bioluminescence requires the interaction of a luciferase enzyme with a small‐molecule luciferin, and its scope has been limited by the mere handful of natural combinations. Herein, we show that mutants of firefly luciferase can discriminate between natural and synthetic substrates in the brains of live mice. When using adeno‐associated viral (AAV) vectors to express luciferases in the brain, we found that mutant luciferases that are inactive or weakly active with d ‐luciferin can light up brightly when treated with the aminoluciferins CycLuc1 and CycLuc2 or their respective FAAH‐sensitive luciferin amides. Further development of selective luciferases promises to expand the power of bioluminescence and allow multiple events to be imaged in the same live animal.     

A Convenient Approach To Synthesize o‐Carborane‐Functionalized Phosphorescent Iridium(III) Complexes for Endocellular Hypoxia Imaging

The structure–property relationship of carborane‐modified iridium(III) complexes was investigated. Firstly, an efficient approach for the synthesis of o‐carborane‐containing pyridine ligands a – f in high yields was developed by utilizing stable and cheap B₁₀H₁₀(Et₄N)₂ as the starting material. By using these ligands, iridium(III) complexes I – VII were efficiently prepared. In combination with DFT calculations, the photophysical and electrochemical properties of these complexes were studied. The hydrophilic nido‐o‐carborane‐based iridium(III) complex VII showed the highest phosphorescence efficiency (abs. =0.48) among known water‐soluble homoleptic cyclometalated iridium(III) complexes and long emission lifetime (τ=1.24 μs) in aqueous solution. Both of them are sensitive to O₂, and thus endocellular hypoxia imaging of complex VII was realized by time‐resolved luminescence imaging (TRLI). This is the first example of applying TRLI in endocellular oxygen detection with a water‐soluble nido‐carborane functionalized iridium(III) complex.     

Polynuclear SmIII Polyamidoamine‐Based Dendrimer: A Single Probe for Combined Visible and Near‐Infrared Live‐Cell Imaging

We report herein the synthesis of a luminescent polynuclear dendritic structure (Sm^III‐G3P‐2,3Nap) in which eight Sm^III ions are sensitized by thirty‐two 2,3‐naphthalimide chromophores. Upon a single excitation wavelength, the dendrimer complex exhibits two types of emission in the visible and in the near‐infrared (NIR) ranges. Sm^III‐G3P‐2,3Nap was non‐cytotoxic after 24 h of incubation and up to 2.5 μM . The ability of the Sm^III‐based probe to be taken up by cells was confirmed by confocal microscopy. Epifluorescence microscopy validated Sm^III‐G3P‐2,3Nap as a versatile probe, capable of performing interchangeably in the visible or NIR for live‐cell imaging. As both emissions are obtained from a single complex, the cytotoxicity and biodistribution are inherently the same. The possibility for discriminating the sharp Sm^III signals from autofluorescence in two spectral ranges increases the reliability of analysis and reduces the probability of artifacts and instrumental errors.     

Cartilage‐Specific Near‐Infrared Fluorophores for Biomedical Imaging

A novel class of near‐infrared fluorescent contrast agents was developed. These agents target cartilage with high specificity and this property is inherent to the chemical structure of the fluorophore. After a single low‐dose intravenous injection and a clearance time of approximately 4 h, these agents bind to all three major types of cartilage (hyaline, elastic, and fibrocartilage) and perform equally well across species. Analysis of the chemical structure similarities revealed a potential pharmacophore for cartilage targeting. Our results lay the foundation for future improvements in tissue engineering, joint surgery, and cartilage‐specific drug development.     

Far‐Red Emitting Fluorescent Dyes for Optical Nanoscopy: Fluorinated Silicon–Rhodamines (SiRF Dyes) and Phosphorylated Oxazines

Far‐red emitting fluorescent dyes for optical microscopy, stimulated emission depletion (STED), and ground‐state depletion (GSDIM) super‐resolution microscopy are presented. Fluorinated silicon–rhodamines (SiRF dyes) and phosphorylated oxazines have absorption and emission maxima at about λ≈660 and 680 nm, respectively, possess high photostability, and large fluorescence quantum yields in water. A high‐yielding synthetic path to introduce three aromatic fluorine atoms and unconventional conjugation/solubilization spacers into the scaffold of a silicon–rhodamine is described. The bathochromic shift in SiRF dyes is achieved without additional fused rings or double bonds. As a result, the molecular size and molecular mass stay quite small (<600 Da). The use of the λ=800 nm STED beam instead of the commonly used one at λ=750–775 nm provides excellent imaging performance and suppresses re‐excitation of SiRF and the oxazine dyes. The photophysical properties and immunofluorescence imaging performance of these new far‐red emitting dyes (photobleaching, optical resolution, and switch‐off behavior) are discussed in detail and compared with those of some well‐established fluorophores with similar spectral properties.     

A Renal‐Clearable Macromolecular Reporter for Near‐Infrared Fluorescence Imaging of Bladder Cancer

Bladder cancer (BC) is a prevalent disease with high morbidity and mortality; however, in vivo optical imaging of BC remains challenging because of the lack of cancer‐specific optical agents with high renal clearance. Herein, a macromolecular reporter (CyP1) was synthesized for real‐time near‐infrared fluorescence (NIRF) imaging and urinalysis of BC in living mice. Because of the high renal clearance (ca. 94 % of the injection dosage at 24 h post‐injection) and its cancer biomarker (APN=aminopeptidase N) specificity, CyP1 can be efficiently transported to the bladder and specially turn on its NIRF signal to report the detection of BC in living mice. Moreover, CyP1 can be used for optical urinalysis, permitting the ex vivo tracking of tumor progression for therapeutic evaluation and easy translation of CyP2 as an in vitro diagnostic assay. This study not only provides new opportunities for non‐invasive diagnosis of BC, but also reveals useful guidelines for the development of molecular reporters for the detection of bladder diseases.     

Covalent Organic Framework for Improving Near‐Infrared Light Induced Fluorescence Imaging through Two‐Photon Induction

Fluorescent materials exhibiting two‐photon induction (TPI) are used for nonlinear optics, bioimaging, and phototherapy. Polymerizations of molecular chromophores to form π‐conjugated structures were hindered by the lack of long‐range ordering in the structure and strong π–π stacking between the chromophores. Reported here is the rational design of a benzothiadiazole‐based covalent organic framework (COF) for promoting TPI and obtaining efficient two‐photon induced fluorescence emissions. Characterization and spectroscopic data revealed that the enhancement in TPI performance is attributed to the donor‐π‐acceptor‐π‐donor configuration and regular intervals of the chromophores, the large π‐conjugation domain, and the long‐range order of COF crystals. The crystalline structure of TPI‐COF attenuates the π–π stacking interactions between the layers, and overcomes aggregation‐caused emission quenching of the chromophores for improving near‐infrared two‐photon induced fluorescence imaging.     

Phosphonated Near‐Infrared Fluorophores for Biomedical Imaging of Bone

The conventional method for creating targeted contrast agents is to conjugate separate targeting and fluorophore domains. A new strategy is based on the incorporation of targeting moieties into the non‐delocalized structure of pentamethine and heptamethine indocyanines. Using the known affinity of phosphonates for bone minerals in a model system, two families of bifunctional molecules that target bone without requiring a traditional bisphosphonate are synthesized. With peak fluorescence emissions at approximately 700 or 800 nm, these molecules can be used for fluorescence‐assisted resection and exploration (FLARE) dual‐channel imaging. Longitudinal FLARE studies in mice demonstrate that phosphonated near‐infrared fluorophores remain stable in bone for over five weeks, and histological analysis confirms their incorporation into the bone matrix. Taken together, a new strategy for creating ultra‐compact, targeted near‐infrared fluorophores for various bioimaging applications is described.     

In Situ Study of the Function of Bacterioruberin in the Dual‐Chromophore Photoreceptor Archaerhodopsin‐4

While certain archaeal ion pumps have been shown to contain two chromophores, retinal and the carotenoid bacterioruberin, the functions of bacterioruberin have not been well explored. To address this research gap, recombinant archaerhodopsin‐4 (aR4), either with retinal only or with both retinal and bacterioruberin chromophores, was successfully expressed together with endogenous lipids in H. salinarum L33 and MPK409 respectively. In situ solid‐state NMR, supported by molecular spectroscopy and functional assays, revealed for the first time that the retinal thermal equilibrium in the dark‐adapted state is modulated by bacterioruberin binding through a cluster of aromatic residues on helix E. Bacterioruberin not only stabilizes the protein trimeric structure but also affects the photocycle kinetics and the ATP formation rate. These new insights may be generalized to other receptors and proteins in which metastable thermal equilibria and functions are perturbed by ligand binding.