Structural Manipulation of Ruthenium(II) Polypyridine Nitrone Complexes to Generate Phosphorogenic Bioorthogonal Reagents for Selective Cellular Labeling

We report a new class of ruthenium(II) polypyridine complexes functionalized with a nitrone group as phosphorogenic bioorthogonal probes. These complexes were very weakly emissive owing to rapid C=N isomerization of the nitrone moiety, but exhibited significant emission enhancement upon strain‐promoted alkyne–nitrone cycloaddition (SPANC) reaction with bicyclo[6.1.0]nonyne (BCN)‐modified substrates. The modification of nitrone with a dicationic ruthenium(II) polypyridine unit at the α‐C‐position and a phenyl ring at the N‐position led to remarkably accelerated reaction kinetics, which are substantially greater (up to ≈278 fold) than those of other acyclic nitrone–BCN systems. Interestingly, the complexes achieved specific cell membrane/cytosol staining upon specific labeling of an exogenous substrate, BCN‐modified decane (BCN‐C10), in live cells. Importantly, the in situ generation of the more lipophilic isoxazoline adduct in the cytoplasm resulted in increased cytotoxicity, highlighting a novel approach to apply the SPANC labeling technique in drug activation.     

Color-Tunable Light-up Bioorthogonal Probes for In Vivo Two-Photon Fluorescence Imaging

Light-up bioorthogonal probes have attracted increasing attention recently due to their capability to directly image diverse biomolecules in living cells without washing steps. The development of bioorthogonal probes with excellent fluorescent properties suitable for in vivo imaging, such as long excitation/emission wavelength, high fluorescence turn-on ratio, and deep penetration, has been rarely reported. Herein, a series of azide-based light-up bioorthogonal probes with tunable colors based on a weak fluorescent 8-aminoquinoline ( AQ ) scaffold were designed and synthesized. The azido quinoline derivatives are able to induce large fluorescence enhancement (up to 1352-fold) after click reaction with alkynes. In addition, the probes could be engineered to exhibit excellent two-photon properties (δ=542 GM at 780 nm) after further introducing different styryl groups into the AQ scaffold. Subsequent detailed bioimaging experiments demonstrated that these versatile probes can be successfully used for live cell/zebrafish imaging without washing steps. Further in vivo two-photon imaging experiments demonstrated that these light-up biorthogonal probe outperformed conventional fluorophores, for example, high signal-to-noise ratio and deep tissue penetration. The design strategy reported in this study is a useful approach to realize diverse high-performance biorthogonal light-up probes for in vivo studying.     

Iridium(III) Anthraquinone Complexes as Two‐Photon Phosphorescence Probes for Mitochondria Imaging and Tracking under Hypoxia

In the present study, four mitochondria‐specific and two‐photon phosphorescence iridium(III) complexes, Ir1 – Ir4 , were developed for mitochondria imaging in hypoxic tumor cells. The iridium(III) complex has two anthraquinone groups that are hypoxia‐sensitive moieties. The phosphorescence of the iridium(III) complex was quenched by the functions of the intramolecular quinone unit, and it was restored through two‐electron bioreduction under hypoxia. When the probes were reduced by reductase to hydroquinone derivative products under hypoxia, a significant enhancement in phosphorescence intensity was observed under one‐ (λ=405 nm) and two‐photon (λ=720 nm) excitation, with a two‐photon absorption cross section of 76–153 GM at λ=720 nm. More importantly, these probes possessed excellent specificity for mitochondria, which allowed imaging and tracking of the mitochondrial morphological changes in a hypoxic environment over a long period of time. Moreover, the probes can visualize hypoxic mitochondria in 3D multicellular spheroids and living zebrafish through two‐photon phosphorescence imaging.     

Tuning the Optical Properties of 2‐Thienylpyridyl Iridium Complexes through Carboranes and Anions

2‐Thienylpyridyl iridium(III) complexes containing an o‐, m‐, or p‐carboranylvinyl‐2,2′‐bipyridine ligand and various counteranions (denoted o ‐ PF₆ , m ‐ BF₄ , m ‐ PF₆ , m ‐ SbF₆ , m ‐ ClO₄ , m ‐ OTf , m ‐ NO₃ , m ‐ BPh₄ , m ‐ F , m ‐ Cl , and p ‐ PF₆ ) were synthesized by using C‐formyl carboranes as starting materials. The solid‐state structures of o ‐ PF₆ , m ‐ PF₆ , m ‐ ClO₄ , and m ‐ BF₄ showed that the cations form twisted cavities in which the anions are fixed by multiple hydrogen bonds. Anion–hydrogen interactions were investigated for nine m‐carborane‐based complexes with different counteranions. All carborane‐based iridium(III) complexes show similar phosphorescence yields in solution but significantly different emission in the solid state. Anion‐exchange titration and theoretical calculations revealed the relationships between structures and optical properties. The size of the anion and C?H ??? X anion–hydrogen bonds strongly influence the phosphorescence quantum yield in the solid state. In particular, the C_car?H ??? X hydrogen bonds between the carboranyl unit and the anion play an important role in solid‐state phosphorescence. Complex p ‐ PF₆ was successfully applied in phosphorescence‐lifetime bioimaging owing to its low toxicity and near‐infrared emission.     

Visible Light‐Triggered Photoswitchable Diarylethene‐Based Iridium(III) Complexes for Imaging Living Cells

A novel diarylethene‐based iridium(III) complex was synthesized as a phosphorescence probe for monitoring living cells. The switchable phosphorescence complex in solution and within living cells was controlled by two distinguishable visible‐light irradiations, which suggests that this complex can be developed as a promising probe with weak photodamage for biological samples.     

Cyclometalated Iridium(III) Complexes as Two‐Photon Phosphorescent Probes for Specific Mitochondrial Dynamics Tracking in Living Cells

Five cyclometalated iridium(III) complexes with 2‐phenylimidazo[4,5‐f][1,10]phenanthroline derivatives ( IrL1 – IrL5 ) were synthesized and developed to image and track mitochondria in living cells under two‐photon (750 nm) excitation, with two‐photon absorption cross‐sections of 48.8–65.5 GM at 750 nm. Confocal microscopy and inductive coupled plasma‐mass spectrometry (ICP‐MS) demonstrated that these complexes selectively accumulate in mitochondria within 5 min, without needing additional reagents for membrane permeabilization, or replacement of the culture medium. In addition, photobleaching experiments and luminescence measurements confirmed the photostability of these complexes under continuous laser irradiation and physiological pH resistance. Moreover, results using 3D multicellular spheroids demonstrate the proficiency of these two‐photon luminescent complexes in deep penetration imaging. Two‐photon excitation using such novel complexes of iridium(III) for exclusive visualization of mitochondria in living cells may substantially enhance practical applications of bioimaging and tracking.     

Red‐ and Green‐Emitting Iridium(III) Complexes for a Dual Barometric and Temperature‐Sensitive Paint

A new dual luminescent sensitive paint for barometric pressure and temperature (T) is presented. The green‐emitting iridium(III) complex [Ir(ppy)₂(carbac)] (ppy=2‐phenylpyridine; carbac=1‐(9H‐carbazol‐9‐yl)‐5,5‐dimethylhexane‐2,4‐dione) was applied as a novel probe for T along with the red‐emitting complex [Ir(btpy)₃], (btpy=2‐(benzo[b]thiophene‐2‐yl)pyridine) which functions as a barometric (in fact oxygen‐sensitive) probe. Both iridium complexes were dissolved in different polymer materials to achieve optimal responses. The probe [Ir(ppy)₂(carbac)] was dispersed in gas‐blocking poly(acrylonitrile) microparticles in order to suppress any quenching of its luminescence by oxygen. The barometric probe [Ir(btpy)₃], in turn, was incorporated in a cellulose acetate butyrate film which exhibits good permeability for oxygen. The effects of temperature on the response of the oxygen probe can be corrected by simultaneous optical determination of T, as the poly(acrylonitrile) microparticles containing the temperature indicator are incorporated into the film. The phosphorescent signals of the probes for T and barometric pressure, respectively, can be separated by optical filters due to the ≈75 nm difference in their emission maxima. The dual sensor is applicable to luminescence lifetime imaging of T and barometric pressure. It is the first luminescent dual sensor material for barometric pressure/T based exclusively on the use of Ir^III complexes in combination with luminescence lifetime imaging.     

Tuning the Cellular Uptake Properties of Luminescent Heterobimetallic Iridium(III)–Ruthenium(II) DNA Imaging Probes

The synthesis of two new luminescent dinuclear Ir^III–Ru^II complexes containing tetrapyrido[3,2‐a:2′,3′‐c:3′′,2′′‐h:2′′′,3′′′‐j]phenazine (tpphz) as the bridging ligand is reported. Unlike many other complexes incorporating cyclometalated Ir^III moieties, these complexes display good water solubility, allowing the first cell‐based study on Ir^III–Ru^II bioprobes to be carried out. Photophysical studies indicate that emission from each complex is from a Ru^II excited state and both complexes display significant in vitro DNA‐binding affinities. Cellular studies show that each complex is rapidly internalised by HeLa cells, in which they function as luminescent nuclear DNA‐imaging agents for confocal microscopy. Furthermore, the uptake and nuclear targeting properties of the complex incorporating cyclometalating 2‐(4‐fluorophenyl)pyridine ligands around its Ir^III centre is enhanced in comparison to the non‐fluorinated analogue, indicating that fluorination may provide a route to promote cell uptake of transition‐metal bioprobes.     

meso‐(2‐Pyridyl)‐boron(III)‐subporphyrin: Perimeter Iridium(III) Coordination

Peripherally metalated porphyrinoids are promising functional π‐systems displaying characteristic optical, electronic, and catalytic properties. In this work, 5‐(2‐pyridyl)‐ and 5,10,15‐tri(2‐pyridyl)‐B^III‐subporphyrins were prepared and used to produce cyclometalated subporphyrins by reactions with [Cp*IrCl₂]₂, which proceeded through an efficient C?H activation to give the corresponding mono‐ and tri‐Ir^III complexes, respectively. While the mono‐Ir^III complex was obtained as a diastereomeric mixture, a C₃‐symmetric tri‐Ir^III complex with the three Cp*‐units all at the concave side was predominantly obtained in a high yield of 90 %, which displays weak NIR phosphorescence even at room temperature in degassed CH₂Cl₂, differently from the mono‐Ir^III complexes.     

Modification of Luminescent Iridium(III) Polypyridine Complexes with Discrete Poly(ethylene glycol) (PEG) Pendants: Synthesis,Emissive Behavior,Intracellular Uptake,and PEGylation Properties

We report the synthesis, characterization, and photophysical properties of a new class of luminescent cyclometalated iridium(III) polypyridine poly(ethylene glycol) (PEG) complexes [Ir(N^C)₂(N^N)](PF₆) (HN^C=Hppy (2‐phenylpyridine), N^N=bpy? CONH? PEG1 (bpy=2,2′‐bipyridine; 1 a ), bpy? CONH? PEG3 ( 1 b ); HN^C=Hpq (2‐phenylquinoline), N^N=bpy? CONH? PEG1 ( 2 a ), bpy? CONH? PEG3 ( 2 b ); HN^C=Hpba (4‐(2‐pyridyl)benzaldehyde), N^N=bpy? CONH? PEG1 ( 3 )) and their PEG‐free counterparts (N^N=bpy? CONH? Et, HN^C=Hppy ( 1 c ); HN^C=Hpq ( 2 c )). The cytotoxicity and cellular uptake of these complexes have been investigated by the MTT assay, ICPMS, laser‐scanning confocal microscopy, and flow cytometry. The results showed that the complexes supported by the water‐soluble PEG can act as biological probes and labels with considerably reduced cytotoxicity. Because the aldehyde groups of complex 3 are reactive toward primary amines, the complex has been utilized as the first luminescent PEGylation reagent. Bovine serum albumin (BSA) and poly(ethyleneimine) (PEI) have been PEGylated with this complex, and the resulting conjugates have been isolated, purified, and their photophysical properties studied. The DNA‐binding and gene‐delivery properties of the luminescent PEI conjugate 3 ‐PEI have also been investigated.     

Cyclometalated Iridium(III) Complexes with Deoxyribose Substituents

Fundamental study of enzymatic nucleoside transport suffers for lack of optical probes that can be tracked noninvasively. Nucleoside transporters are integral membrane glycoproteins that mediate the salvage of nucleosides and their passage across cell membranes. The substrate recognition site is the deoxyribose sugar, often with little distinction among nucleobases. Reported here are nucleoside analogues in which emissive, cyclometalated iridium(III) complexes are “clicked” to C‐1 of deoxyribose in place of canonical nucleobases. The resulting complexes show visible luminescence at room temperature and 77 K with microsecond‐length triplet lifetimes. A representative complex is crystallographically characterized. Transport and luminescence are demonstrated in cultured human carcinoma (KB3‐1) cells.     

Development of a 18F‐Labeled Tetrazine with Favorable Pharmacokinetics for Bioorthogonal PET Imaging

A low‐molecular‐weight ¹⁸F‐labeled tetrazine derivative was developed as a highly versatile tool for bioorthogonal PET imaging. Prosthetic groups and undesired carrying of ¹⁸F through additional steps were evaded by direct ¹⁸F‐fluorination of an appropriate tetrazine precursor. Reaction kinetics of the cycloaddition with trans‐cyclooctenes were investigated by applying quantum chemical calculations and stopped‐flow measurements in human plasma; the results indicated that the labeled tetrazine is suitable as a bioorthogonal probe for the imaging of dienophile‐tagged (bio)molecules. In vitro and in vivo investigations revealed high stability and PET/MRI in mice showed fast homogeneous biodistribution of the ¹⁸F‐labeled tetrazine that also passes the blood–brain barrier. An in vivo click experiment confirmed the bioorthogonal behavior of this novel tetrazine probe. Due to favorable chemical and pharmacokinetic properties this bioorthogonal agent should find application in bioimaging and biomedical research.     

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.     

Rhodium(III)‐ and Iridium(III)‐Catalyzed C7 Alkylation of Indolines with Diazo Compounds

A Rh^III‐catalyzed procedure for the C7‐selective C?H alkylation of various indolines with α‐diazo compounds at room temperature is reported. The advantages of this process are: 1) simple, mild, and pH‐neutral reaction conditions, 2) broad substrate scope, 3) complete regioselectivity, 4) no need for an external oxidant, and 5) N₂ as the sole byproduct. Furthermore, alkylation and bis‐alkylation of carbazoles at the C1 and C8 positions have also been developed. More significantly, for the first time, a successful Ir^III‐catalyzed intermolecular insertion of arene C?H bonds into α‐diazo compounds is reported.     

Probing the Anticancer Mechanism of (−)‐Ainsliatrimer A through Diverted Total Synthesis and Bioorthogonal Ligation

Herein, we report an efficient approach for exploring the novel anticancer mechanism of (?)‐ainsliatrimer A, a structurally complex and unique trimeric sesquiterpenoid, through a combined strategy of diverted total synthesis (DTS) and bioorthogonal ligation (TQ ligation), which allowed us to visualize the subcellular localization of this natural product in live cells. Further biochemical studies facilitated by pretarget imaging revealed that PPARγ, a nucleus receptor, was a functional cellular target of ainsliatrimer A. We also confirmed that the anticancer activity of ainsliatrimer A was caused by the activation of PPARγ.     

A Dansyl Amide N-Oxide Fluorogenic Probe Based on a Bioorthogonal Decaging Reaction

A smart fluorescence “turn-on” probe which contained a dansyl amide fluorophore and an N-oxide group was designed based on the bioorthogonal decaging reaction between N-oxide and the boron reagent. The reaction proceeds in a rapid kinetics (k₂=57.1±2.5 m ⁻¹ s⁻¹), and the resulting reduction product showcases prominent fluorescence enhancement (up to 72-fold). Time dependent density functional theoretical (TD-DFT) calculation revealed that the process of photoinduced electron transfer (PET) from the N-oxide moiety to the dansyl amide fluorophore accounts for the quenching mechanism of N-oxide. This probe also showed high selectivity over various nucleophilic amino acids and good biocompatibility in physiological conditions. The successful application of the probe in HaloTag protein labeling and HepG2 live-cell imaging proves it a valuable tool for visualization of biomolecules.