Rapid Peptide Cyclization Inspired by the Modular Logic of Nonribosomal Peptide Synthetases.

Nonribosomal cyclic peptides (NRcPs) are structurally complex natural products and a vital pool of therapeutics, particularly antibiotics. Their structural diversity arises from the ability of the multidomain enzyme assembly lines, nonribosomal peptide synthetases (NRPSs), to utilize bespoke nonproteinogenic amino acids, modify the linear peptide during elongation, and catalyze an array of cyclization modes, e.g., head to tail, side chain to tail. The study and drug development of NRcPs are often limited by a lack of easy synthetic access to NRcPs and their analogues, with selective macrolactamization being a major bottleneck. Herein, we report a generally applicable chemical macrocyclization method of unprecedented speed and selectivity. Inspired by biosynthetic cyclization, it combines the deprotected linear biosynthetic precursor peptide sequence with a highly reactive C-terminus to produce NRcPs and analogues in minutes. The method was applied to several NRcPs of varying sequences, ring sizes, and cyclization modes including rufomycin, colistin, and gramicidin S with comparable success. We thus demonstrate that the linear order of modules in NRPS enzymes that determines peptide sequence encodes the key structural information to produce peptides conformationally biased toward macrocyclization. To fully exploit this conformational bias synthetically, a highly reactive C-terminal acyl azide is also required, alongside carefully balanced pH and solvent conditions. This allows for consistent, facile cyclization of exceptional speed, selectivity, and atom efficiency. This exciting macrolactamization method represents a new enabling technology for the biosynthetic study of NRcPs and their development as therapeutics.

Antibacterial activity of Au(I), Pt(II), and Ir(III) biotin conjugates prepared by the iClick reaction: influence of the metal coordination sphere on the biological activity.

A series of biotin-functionalized transition metal complexes was prepared by iClick reaction from the corresponding azido complexes with a novel alkyne-functionalized biotin derivative ([Au(triazolatoR,R')(PPh3)], [Pt(dpb)(triazolatoR,R')], [Pt(triazolatoR,R')(terpy)]PF6, and [Ir(ppy)(triazolatoR,R')(terpy)]PF6 with dpb = 1,3-di(2-pyridyl)benzene, ppy = 2-phenylpyridine, and terpy = 2,2':6',2''-terpyridine and R = C6H5, R' = biotin). The complexes were compared to reference compounds lacking the biotin moiety. The binding affinity toward avidin and streptavidin was evaluated with the HABA assay as well as isothermal titration calorimetry (ITC). All compounds exhibit the same binding stoichiometry of complex-to-avidin of 4:1, but the ITC results show that the octahedral Ir(III) compound exhibits a higher binding affinity than the square-planar Pt(II) complex. The antibacterial activity of the compounds was evaluated on a series of Gram-negative and Gram-positive bacterial strains. In particular, the neutral Au(I) and Pt(II) complexes showed significant antibacterial activity against Staphylococcus aureus and Enterococcus faecium at very low micromolar concentrations. The cytotoxicity against a range of eukaryotic cell lines was studied and revealed that the octahedral Ir(III) complex was non-toxic, while the square-planar Pt(II) and linear Au(I) complexes displayed non-selective micromolar activity.

Recent Advances in Metal Complexes for Antimicrobial Photodynamic Therapy.

Antimicrobial resistance (AMR) is a growing global problem with more than 1 million deaths due to AMR infection in 2019 alone. New and innovative therapeutics are required to overcome this challenge. Antimicrobial photodynamic therapy (aPDT) is a rapidly growing area of research poised to provide much needed help in the fight against AMR. aPDT works by administering a photosensitizer (PS) that is activated only when irradiated with light, allowing high spatiotemporal control and selectivity. The PS typically generates reactive oxygen species (ROS), which can damage a variety of key biological targets, potentially circumventing existing resistance mechanisms. Metal complexes are well known to display excellent optoelectronic properties, and recent focus has begun to shift towards their application in tackling microbial infections. Herein, we review the last five years of progress in the emerging field of small-molecule metal complex PSs for aPDT.

A self-assembled Ru-Pt metallacage as a lysosome-targeting photosensitizer for 2-photon photodynamic therapy.

Photodynamic therapy (PDT) is a treatment procedure that relies on cytotoxic reactive oxygen species (ROS) generated by the light activation of a photosensitizer. The photophysical and biological properties of photosensitizers are vital for the therapeutic outcome of PDT. In this work a 2D rhomboidal metallacycle and a 3D octahedral metallacage were designed and synthesized via the coordination-driven self-assembly of a Ru(II)-based photosensitizer and complementary Pt(II)-based building blocks. The metallacage showed deep-red luminescence, a large 2-photon absorption cross-section, and highly efficient ROS generation. The metallacage was encapsulated into an amphiphilic block copolymer to form nanoparticles to encourage cell uptake and localization. Upon internalization into cells, the nanoparticles selectively accumulate in the lysosomes, a favorable location for PDT. The nanoparticles are almost nontoxic in the dark, and can efficiently destroy tumor cells via the generation of ROS in the lysosomes under 2-photon near-infrared light irradiation. The superb PDT efficacy of the metallacage-containing nanoparticles was further validated by studies on 3D multicellular spheroids (MCS) and in vivo studies on A549 tumor-bearing mice.

Heterometallic Ru-Pt metallacycle for two-photon photodynamic therapy.

As an effective and noninvasive treatment of various diseases, photodynamic therapy (PTD) relies on the combination of light, a photosensitizer, and oxygen to generate cytotoxic reactive oxygen species that can damage malignant tissue. Much attention has been paid to covalent modifications of the photosensitizers to improve their photophysical properties and to optimize the pathway of the photosensitizers interacting with cells within the target tissue. Herein we report the design and synthesis of a supramolecular heterometallic Ru-Pt metallacycle via coordination-driven self-assembly. While inheriting the excellent photostability and two-photon absorption characteristics of the Ru(II) polypyridyl precursor, the metallacycle also exhibits red-shifted luminescence to the near-infrared region, a larger two-photon absorption cross-section, and higher singlet oxygen generation efficiency, making it an excellent candidate as a photosensitizer for PTD. Cellular studies reveal that the metallacycle selectively accumulates in mitochondria and nuclei upon internalization. As a result, singlet oxygen generated by photoexcitation of the metallacycle can efficiently trigger cell death via the simultaneous damage to mitochondrial function and intranuclear DNA. In vivo studies on tumor-bearing mice show that the metallacycle can efficiently inhibit tumor growth under a low light dose with minimal side effects. The supramolecular approach presented in this work provides a paradigm for the development of PDT agents with high efficacy.

Supramolecular Assembly of An Organoplatinum(II) Complex with Ratiometric Dual Emission for Two-Photon Bioimaging.

The organoplatinum(II) complex [Pt(C^N^N)(Cl)] (C^N^N=5,6-diphenyl-2,2'-bipyridine, Pt1) can assemble into nanoaggregates via π-π stacking and complementary hydrogen bonds, rather than Pt-Pt interactions. Pt1 exhibits ratiometric dual emission, including rare blue emission (λem =445 nm) and assembly-induced yellow emission (λem =573 nm), under one- and two-photon excitation. Pt1 displays blue emission in cells with an intact membrane due to its low cellular uptake. In cells where the membrane is disrupted, uptake of the complex is increased and at higher concentrations yellow emission is observed. The ratio of yellow to blue emission shows a linear relationship to the loss of cell membrane integrity. Pt1 is, to our knowledge, the first example of an assembly-induced two-photon ratiometric dual emission organoplatinum complex. The excellent and unique characteristics of the complex enabled its use for the tracking of cell apoptosis, necrosis, and the inflammation process in zebrafish.

An ER-Targeting Iridium(III) Complex That Induces Immunogenic Cell Death in Non-Small-Cell Lung Cancer.

Immunogenic cell death (ICD) is a vital component of therapeutically induced anti-tumor immunity. An iridium(III) complex (Ir1), containing an N,N-bis(2-chloroethyl)-azane derivate, as an endoplasmic reticulum-localized ICD inducer for non-small cell lung cancer (NSCLC) is reported. The characteristic discharge of damage-associated molecular patterns (DAMPs), that is, cell surface exposure of calreticulin (CRT), extracellular exclusion of high mobility group box 1 (HMGB1), and ATP, were generated by Ir1 in A549 lung cancer cells, accompanied by an increase in endoplasmic reticulum stress and reactive oxygen species (ROS). The vaccination of immunocompetent mice with Ir1-treated dying cells elicited an antitumor CD8+ T cell response and Foxp3+ T cell depletion, which eventually resulted in long-acting anti-tumor immunity by the activation of ICD in lung cancer cells. Ir1 is the first Ir-based complex that is capable of developing an immunomodulatory response by immunogenic cell death.

Lysosome-Targeting Iridium(III) Probe with Near-Infrared Emission for the Visualization of NO/O2•- Crosstalk via In Vivo Peroxynitrite Imaging.

Nitric oxide (NO) and superoxide anions (O2•-) are two noteworthy reactive species implicated in various physiological and pathological processes, such as ROS-induced lysosomal cell death. The interaction ("crosstalk") between them may form a new mediator peroxynitrite (ONOO-) which has implications for cancer, diabetes, Alzheimer's disease, and liver-damage. It is therefore essential to investigate lysosomal NO/O2•- crosstalk in vivo through ONOO--responsive molecular tools in order to fully comprehend the physiological and pathological mechanisms involved. In this study, a lysosome-targeting iridium(III) complex, Ir-NIR, has been investigated as a near-infrared (NIR) phosphorescent probe for visualizing NO/O2•- crosstalk by the phosphorescent detection of endogenous ONOO- levels in vivo. Ir-NIR exhibits a rapid (within 200 s), highly sensitive, and approximately 100-fold enhanced response to ONOO- in phosphorescence intensity. Thus, these characteristics, coupled with good cell permeability and low cytotoxicity, enable the probe to be used to detect intracellular ONOO- living organisms both in vitro and in vivo.

FerriIridium: A Lysosome-Targeting Iron(III)-Activated Iridium(III) Prodrug for Chemotherapy in Gastric Cancer Cells.

Reported is the FeIII -activated lysosome-targeting prodrug FerriIridium for gastric cancer theranostics. It contains a meta-imino catechol group that can selectively bond to, and be oxidized by, free FeIII inside the cell. Subsequent oxidative rearrangement releases FeII and hydrolyses the amine bond under acidic conditions, forming an aminobipyridyl Ir complex and 2-hydroxybenzoquinone. Thus, FeII catalyzes the Fenton reaction, transforming hydrogen peroxide into hydroxyl radicals, the benzoquinone compounds interfere with the respiratory chain, and conversion of the prodrug into the Ir complex leads to an increase in phosphorescence and toxicity. These properties, combined with the high FeIII content and acidity of cancer cells, make FerriIridium a selective and efficient theranostic agent (IC50 =9.22 µm for AGS cells vs. >200 µm for LO2 cells). FerriIridium is the first metal-based compound that has been developed for chemotherapy using FeIII to enhance both selectivity and potency.

A Mitochondrion-Localized Two-Photon Photosensitizer Generating Carbon Radicals Against Hypoxic Tumors.

The efficacy of photodynamic therapy is typically reliant on the local concentration and diffusion of oxygen. Due to the hypoxic microenvironment found in solid tumors, oxygen-independent photosensitizers are in great demand for cancer therapy. We herein report an iridium(III) anthraquinone complex as a mitochondrion-localized carbon-radical initiator. Its emission is turned on under hypoxic conditions after reduction by reductase. Furthermore, its two-photon excitation properties (λex =730 nm) are highly desirable for imaging. Upon irradiation, the reduced form of the complex generates carbon radicals, leading to a loss of mitochondrial membrane potential and cell death (IC50light =2.1 µm, IC50dark =58.2 µm, PI=27.7). The efficacy of the complex as a PDT agent was also demonstrated under hypoxic conditions in vivo. To the best of our knowledge, it is the first metal-complex-based theranostic agent which can generate carbon radicals for oxygen-independent two-photon photodynamic therapy.

Necroptosis Induced by Ruthenium(II) Complexes as Dual Catalytic Inhibitors of Topoisomerase I/II.

Inducing necroptosis in cancer cells is an effective approach to circumvent drug-resistance. Metal-based triggers have, however, rarely been reported. Ruthenium(II) complexes containing 1,1-(pyrazin-2-yl)pyreno[4,5-e][1,2,4]triazine were developed with a series of different ancillary ligands (Ru1-7). The combination of the main ligand with bipyridyl and phenylpyridyl ligands endows Ru7 with superior nucleus-targeting properties. As a rare dual catalytic inhibitor, Ru7 effectively inhibits the endogenous activities of topoisomerase (topo) I and II and kills cancer cells by necroptosis. The cell signaling pathway from topo inhibition to necroptosis was elucidated. Furthermore, Ru7 displays significant antitumor activity against drug-resistant cancer cells in vivo. To the best of our knowledge, Ru7 is the first Ru-based necroptosis-inducing chemotherapeutic agent.

Rational Design of Cyclometalated Iridium(III) Complexes for Three-Photon Phosphorescence Bioimaging.

Compared to 2PE (two-photon excitation) microscopy, 3PE microscopy has superior spatial resolution, deeper tissue penetration, and less defocused interference. The design of suitable agents with a large Stokes shift, good three-photon absorption (3PA), subcellular targeting, and fluorescence lifetime imaging (FLIM) properties, is challenging. Now, two IrIII complexes (3PAIr1 and 3PAIr2) were developed as efficient three-photon phosphorescence (3PP) agents. Calculations reveal that the introduction of a new group to the molecular scaffold confers a quadruple promotion in three-photon transition probability. Confocal and lifetime imaging of mitochondria using IrIII complexes as 3PP agents is shown. The complexes exhibit low working concentration (50 nm), fast uptake (5 min), and low threshold for three-photon excitation power (0.5 mW at 980 nm). The impressive tissue penetration depth (ca. 450 µm) allowed the 3D imaging and reconstruction of brain vasculature from a living specimen.

Bimodal Visualization of Endogenous Nitric Oxide in Lysosomes with a Two-Photon Iridium(III) Phosphorescent Probe.

Nitric oxide (NO) is a fundamental signaling molecule that shows complex effects on the catabolic autophagy process, which is closely linked with lysosomal function. In this study, a new lysosome-targeted, pH-independent, and two-photon phosphorescent iridium(III) complex, Ir-BPDA, has been investigated for endogenous NO detection and imaging. The rational design of the probe, as the addition of the morpholine moieties and the substitution of a benzyl group in the amino group in Ir-BPDA, facilitates its accumulation in lysosomes and makes the reaction product with NO, Ir-BPDA-NO, insusceptible in its phosphorescence intensity and lifetime against pH changes (pH 4-10), well suited for lysosomal NO detection (pH 4-6). Furthermore, Ir-BPDA exhibits a fast and 50-fold response to NO in phosphorescence intensity and a two-photon cross-section as high as 60 GM after the reaction, as well as a notably increased phosphorescence lifetime from 200.1 to 619.6 ns. Thus, accompanied by its photostability, Ir-BPDA enabled the detection of NO in the lipopolysaccharide-stimulated macrophages and zebrafish model, revealing the endogenous lysosomal NO distribution during inflammation in vivo by means of both TPM and PLIM imaging techniques.

Interfering with DNA High-Order Structures using Chiral Ruthenium(II) Complexes.

In this work, it was found that DNA can undergo B-Z transformational changes and compaction in the presence of DNA intercalators such as ruthenium(II) polypyridyl complexes. The link between B-Z transition and condensation is weak but can be strengthened under certain circumstances with slight alterations to the structures of the ruthenium(II) complexes. Here, following on from previous research, this work reports a series of ruthenium(II) complexes with imidazophenanthroline ligands, which vary in size and planarity. The complexes exhibit distinct effects on DNA structures, ranging from little impact to the transformation of DNA secondary structures to the formation of higher-order DNA structures. Further studies on DNA morphological changes induced by chiral ruthenium(II) complexes are observed by atomic force microscopy and transmission electron microscopy.

Synthesis and Characterization of a Series of Bis-homoleptic Cycloruthenates with Terdentate Ligands as a Family of Panchromatic Dyes.

A series of six homoleptic bis-cyclometalated ruthenium complexes, Ru(N^N^C)2, is reported where N^N^C is a 6-(2,4-difluoro-3-R3-phenyl)-4-R2-4'-R1-2,2'-bipyridine with R3 = -H or -CF3 and R2 and R1 = -COOEt or -CF3. An effective synthesis of the ligands and the complexes is described. The UV-visible absorption studies demonstrate that these complexes are panchromatic dyes absorbing up to 900 nm. Importantly, the onset of absorption depends only on the substitution on the metalated phenyl, whereas the intensity of absorption throughout the spectra is a function of substituents on both the phenyl and the bipyridine moieties. The same trend is observed in electrochemistry as the redox gap depends only on the substitution on the metalated phenyl, whereas the oxidation and reduction potentials are a function of substituents on both the phenyl and the bipyridine moieties. Preliminary tests as sensitizer for dye-sensitized solar cells demonstrate that the number of anchoring groups on the dye has a major influence on the device efficiency.

Ruthenium(II) complexes coordinated to graphitic carbon nitride: Oxygen self-sufficient photosensitizers which produce multiple ROS for photodynamic therapy in hypoxia.

The photodynamic therapy (PDT) of cancer is limited by tumor hypoxia as PDT efficiency depends on O2 concentration. A novel oxygen self-sufficient photosensitizer (Ru-g-C3N4) was therefore designed and synthesized via a facile one-pot method in order to overcome tumor hypoxia-induced PDT resistance. The photosensitizer is based on [Ru(bpy)2]2+ coordinated to g-C3N4 nanosheets by Ru-N bonding. Compared to pure g-C3N4, the resulting nanosheets exhibit increased water solubility, stronger visible light absorption, and enhanced biocompatibility. Once Ru-g-C3N4 is taken up by hypoxic tumor cells and exposed to visible light, the nanosheets not only catalyze the decomposition of H2O2 and H2O to generate O2, but also catalyze H2O2 and O2 concurrently to produce multiple ROS (•OH, •O2-, and 1O2). In addition, Ru-g-C3N4 affords luminescence imaging, while continuously generating O2 to alleviate hypoxia greatly improving PDT efficacy. To the best of our knowledge, this oxygen self-sufficient photosensitizer produced via grafting a metal complex onto g-C3N4 is the first of its type to be reported.

Nano-assembly of ruthenium(II) photosensitizers for endogenous glutathione depletion and enhanced two-photon photodynamic therapy.

Photodynamic therapy (PDT) is a promising noninvasive cancer treatment. PDT in the clinic faces several hurdles due to the unique tumor environment, a feature of which is high levels of glutathione (GSH). An excess amount of GSH consumes reactive oxygen species (ROS) generated by photosensitizers (PSs), reducing PDT efficiency. Herein, nano-photosensitizers (RuS1 NPs and RuS2 NPs) are reported. These consist of ruthenium complexes joined by disulfide bonds forming GSH sensitive polymer nanoparticles. The NPs achieve enhanced uptake compared to their constituent monomers. Inside cancer cells, high levels of GSH break the S-S bonds releasing PS molecules in the cell. The level of GSH is also then reduced leading to excellent PDT activity. Furthermore, RuS2 NPs functionalized with tumor targeting hyaluronic acid (HA@RuS2 NPs) assessed in vivo were highly effective with minimal side effects. To the best of our knowledge, RuS NPs are the first metal complex-based nano-assembled photosensitizers which exhibit enhanced specificity and consume endogenous GSH simultaneously, thus achieving excellent two-photon PDT efficiency in vitro and in vivo.

A mitochondria-targeting magnetothermogenic nanozyme for magnet-induced synergistic cancer therapy.

Magnetic hyperthermia therapy (MHT) and chemodynamic therapy (CDT) are non-invasive in situ treatments without depth limitations and with minimum adverse effects on surrounding healthy tissue. We herein report a mitochondria-targeting magnetothermogenic nanozyme (Ir@MnFe2O4 NPs) for highly efficient cancer therapy. An iridium(III) complex (Ir) acts as a mitochondria-targeting agent on the surface of MnFe2O4 NPs. On exposure to an alternating magnetic field (AMF), the Ir@MnFe2O4 NPs induce a localized increase in temperature causing mitochondrial damage (MHT effect). Meanwhile glutathione (GSH) reduces Fe(III) to Fe(II) on the NPs surface, which in turn catalyzes the conversion of H2O2 to cytotoxic •OH (CDT effect). The depletion of GSH (a •OH scavenger) increases CDT efficacy, while the localized increase in temperature increases the rate of conversion of both Fe(III) to Fe(II) and H2O2 to •OH further enhancing the CDT effect. In addition, the disruption of cellular redox homeostasis due to CDT, leads to greater sensitivity of the cell towards MHT. This nanoplatform integrates these excellent therapeutic properties, with two-photon microscopy (TPM) (demonstrated in vitro) and magnetic resonance imaging (MRI) (demonstrated in vivo) to enable the precise and effective treatment of cancer.

Metal complexes for mitochondrial bioimaging.

Mitochondria are essential organelles in eukaryotic cells, containing various signaling molecules and important enzymes associated with cell growth, death, and proliferation. The visualization of mitochondria and their biochemistry with confocal microscopy, fluorescence (phosphorescence) lifetime microscopy (FLIM, PLIM), and super-resolution microscopy has therefore been of great interest in recent years. In particular, transition metal complexes have emerged as excellent mitochondria-targeting probes, due to their high photostabilities, large Stokes shifts, tunable chemical structures and long luminescence lifetimes. In this review, we focus on platinum, ruthenium and iridium complexes, and their application as detectors of micro-environmental alterations as well as for the imaging of signaling molecules inside mitochondria.

Correction: Mitochondria-targeted Ir@AuNRs as bifunctional therapeutic agents for hypoxia imaging and photothermal therapy.

Correction for 'Mitochondria-targeted Ir@AuNRs as bifunctional therapeutic agents for hypoxia imaging and photothermal therapy' by Libing Ke et al., Chem. Commun., 2019, 55, 10273-10276.