Facile Method to Obtain Functionalised η6-Bound Arenes in Ru(II) and Os(II) Half-Sandwich Complexes.

Half-sandwich Ru(II)- and Os(II)-arene complexes have great potential for catalytic and biological applications. The possibility of fine-tuning their chemical reactivity by including modifications in the ligands around the metal adds to their many advantages. However, structural modifications at the n6-bound arene have had significant synthetic limitations, particularly in the design of Os(II)-tethered complexes. For the first time, we have employed a practical C(sp3)-C(sp2) coupling to obtain 28 new Ru(II) and Os(II) η6-arene half-sandwich complexes with a wide variety of arene functionalities, including those that enable the formation of tether rings, such as quinoline and coumarin. The introduction of novel functional groups at the arene in Ru(II)- and Os(II) half-sandwich complexes can broaden the synthetic scope of this type of organometallic complexes, and help to take full advantage of their structural diversity, especially in intracellular catalysis.

OsO2 as the Contrast-Generating Chemical Species of Osmium-Stained Biological Tissues in Electron Microscopy.

Electron imaging of biological samples stained with heavy metals has enabled visualization of subcellular structures critical in chemical-, structural-, and neuro-biology. In particular, osmium tetroxide OsO4 has been widely adopted for selective lipid imaging. Despite the ubiquity of its use, the osmium speciation in lipid membranes and the process for contrast generation in electron microscopy (EM) have continued to be open questions, limiting efforts to improve staining protocols and therefore high-resolution nanoscale imaging of biological samples. Following our recent success using photoemission electron microscopy (PEEM) to image mouse brain tissues with synaptic resolution, we have used PEEM to determine the nanoscale electronic structure of Os-stained biological samples. Os(IV), in the form of OsO2, generates nanoaggregates in lipid membranes, leading to a strong spatial variation in the electronic structure and electron density of states. OsO2 has a metallic electronic structure that drastically increases the electron density of states near the Fermi level. Depositing metallic OsO2 in lipid membranes allows for strongly enhanced EM signals and conductivity of biological materials. The identification of the chemical species and understanding of the membrane contrast mechanism of Os-stained biological specimens provides a new opportunity for the development of staining protocols for high-resolution, high-contrast EM imaging.

Laminarin-modulated osmium nanozymes with high substrate-affinity and selective peroxidase-like behavior engineered colorimetric assay for hydroxyl radical scavenging capacity estimation.

Hydroxyl radical (·OH) scavenging capacity (HOSC) estimation is essential for evaluating antioxidants, natural extracts, or drugs against clinical diseases. While nanozymes offer advantages in related applications, they still face limitations in activity and selectivity. In response, this work showcases the fabrication of laminarin-modulated osmium (laminarin-Os) nanoclusters (1.45 ± 0.05 nm), functioning as peroxidase-like nanozymes within a colorimetric assay tailored for rational HOSC estimation. This study validates both the characterization and remarkable stability of laminarin-Os. By leveraging the abundant surface negative charges of laminarin-Os and the surface hydroxyls of laminarin, oxidation reactions are facilitated, augmenting laminarin-Os's affinity for 3,3',5,5'-tetramethylbenzidine (TMB) (KM = 0.04 mM). This enables the laminarin-Os-based colorimetric assay to respond to ·OH more effectively than citrate-, albumin-, or other polysaccharides-based Os. In addition, experimental results also validate the selective peroxidase-like behavior of laminarin-Os under acidic conditions. Antioxidants like ascorbic acid, glutathione, tannic acid, and cysteine inhibit absorbance at 652 nm in the colorimetric platform using laminarin-Os's peroxidase-like activity. Compared with commercial kits, this assay demonstrates superior sensitivity (e.g., responds to ascorbic acid 0.01-0.075 mM, glutathione 1-15 µg/mL, tannic acid 0.5-5 µM, and monoammonium glycyrrhizinate cysteine 1.06-10.63 µM) and HOSC testing for glutathione, tannic acid, and monoammonium glycyrrhizinate cysteine. Overall, this study introduces a novel Os nanozyme with exceptional TMB affinity and ·OH selectivity, paving the way for HOSC estimation in biomedical research, pharmaceutical analysis, drug quality control, and beyond.

In-cell Catalysis by Tethered Organo-Osmium Complexes Generates Selectivity for Breast Cancer Cells.

Anticancer agents that exhibit catalytic mechanisms of action offer a unique multi-targeting strategy to overcome drug resistance. Nonetheless, many in-cell catalysts in development are hindered by deactivation by endogenous nucleophiles. We have synthesised a highly potent, stable Os-based 16-electron half-sandwich ('piano stool') catalyst by introducing a permanent covalent tether between the arene and chelated diamine ligand. This catalyst exhibits antiproliferative activity comparable to the clinical drug cisplatin towards triple-negative breast cancer cells and can overcome tamoxifen resistance. Speciation experiments revealed Os to be almost exclusively albumin-bound in the extracellular medium, while cellular accumulation studies identified an energy-dependent, protein-mediated Os accumulation pathway, consistent with albumin-mediated uptake. Importantly, the tethered Os complex was active for in-cell transfer hydrogenation catalysis, initiated by co-administration of a non-toxic dose of sodium formate as a source of hydride, indicating that the Os catalyst is delivered to the cytosol of cancer cells intact. The mechanism of action involves the generation of reactive oxygen species (ROS), thus exploiting the inherent redox vulnerability of cancer cells, accompanied by selectivity for cancerous cells over non-tumorigenic cells.

Emulsion Liquid Membranes Based on Os-NP/n-Decanol or n-Dodecanol Nanodispersions for p-Nitrophenol Reduction.

Membrane materials with osmium nanoparticles have been recently reported for bulk membranes and supported composite membrane systems. In the present paper, a catalytic material based on osmium dispersed in n-decanol (nD) or n-dodecanol (nDD) is presented, which also works as an emulsion membrane. The hydrogenation of p-nitrophenol (PNP) is carried out in a reaction and separation column in which an emulsion in the acid-receiving phase is dispersed in an osmium nanodispersion in n-alcohols. The variables of the PNP conversion process and p-aminophenol (PAP) transport are as follows: the nature of the membrane alcohol, the flow regime, the pH difference between the source and receiving phases and the number of operating cycles. The conversion results are in all cases better for nD than nDD. The counter-current flow regime is superior to the co-current flow. Increasing the pH difference between the source and receiving phases amplifies the process. The number of operating cycles is limited to five, after which the regeneration of the membrane dispersion is required. The apparent catalytic rate constant (kapp) of the new catalytic material based on the emulsion membrane with the nanodispersion of osmium nanoparticles (0.1 × 10-3 s-1 for n-dodecanol and 0.9 × 10-3 s-1 for n-decanol) is lower by an order of magnitude compared to those based on adsorption on catalysts from the platinum metal group. The advantage of the tested membrane catalytic material is that it extracts p-aminophenol in the acid-receiving phase.

A brief history of the Feulgen reaction.

One hundred years ago, Robert Feulgen published a landmark paper in which he described the first method to stain DNA in cells and tissues. Although a century has passed since the discovery by Feulgen and Rossenbeck, the chemical reaction still exerts an important influence in current histochemical studies. Its contribution in diverse fields, spanning from biomedicine to plant biology, has paved the way for the most significant studies that constitute our current knowledge. The possibility to specifically explore the DNA in cell nuclei while quantifying its content makes it a contemporary and timeless method. Indeed, many histocytochemical studies following the 1924 paper have led to a deep understanding of genome organization in general as well as several specific mechanisms (e.g. DNA duplication or tumour pathology) that, nowadays, constitute some of the most fundamental pillars in biological investigations. In this review, we discuss the chemistry and application of the Feulgen reaction to both light and electron microscopy.

Ab initio multireference calculation of electronic spectra of the osmium complexes, [Os(bpy) 3 ] 2 + and [Os(phen) 3 ] 2 + .

The spin-orbit coupling corrected absorption spectra of osmium complexes, [Os(bpy) 3 ] 2 + and [Os(phen) 3 ] 2 + , were calculated by using ab initio multireference perturbation method (NEVPT2) with relativistic effects taken into account throughout ZORA approximation and corresponding all-electron basis sets. For the same purpose, the time-dependent DFT techniques were used. A very good agreement between NEVPT2 and experimental spectra should be highlighted, especially for the MLCT transitions that occur in visible and near-UV regions ( 16 , 000 - 33 , 000 cm - 1 ). Moreover, the present study offers description of excited states of titled osmium complexes and their spectra interpretation using molecular orbitals.

Chemotherapeutic Activities of New η6-p-Cymene Ruthenium(II) and Osmium(II) Complexes with Chelating SS and Tridentate SNS Ligands.

A series of new chelating bidentate (SS) alkylimidazole-2-thione-Ru(II)/Os(II) complexes (3ai, 3aii, 3aiii, 3bii/4aiii, 4bi, 4bii), and the tridentate (SNS) pyridine-2,6-diylimidazole-2-thione-Ru(II)/Os(II) complexes (5bi, 5civ/6bi, 6ci, 6civ) in the forms [MII(cym)(L)Cl]PF6 and [MII(cym)(L)]PF6 (M = Ru or Os, cym = η6-p-cymene, and L = heterocyclic derivatives of thiourea) respectively, were successfully synthesized. Spectroscopic and analytical methods were used to characterize the complexes and their ligands. Solid-state single-crystal X-ray diffraction analyses revealed a "piano-stool" geometry around the Ru(II) or Os(II) centers in the respective complexes. The complexes were investigated for in vitro chemotherapeutic activities against human cervical carcinoma (HeLa) and the non-cancerous cell line (Hek293) using the MTT assay. The compounds 3aii, 5civ, 5bi, 4aiii, 6ci, 6civ, and the reference drug, 5-fluorouracil were found to be selective toward the tumor cells; the compounds 3ai, 3aiii, 3bii, 4bi, 4bii, and 6bi, which were found not to be selective between normal and tumor cell lines. The IC50 value of the tridentate half-sandwich complex 5bi (86 ± 9 µM) showed comparable anti-proliferative activity with the referenced commercial anti-cancer drug, 5-fluorouracil (87 ± 15 µM). The pincer (SNS) osmium complexes 6ci (36 ± 10 µM) and 6civ (40 ± 4 µM) were twice as effective as the reference drug 5-fluorouracil at the respective dose concentrations. However, the analogous pincer (SNS) ruthenium complex 5civ was ineffective and did not show anti-proliferative activity, even at a higher concentration of 147 ± 1 µM. These findings imply that the higher stability of the chelating (SS) and the pincer (SNS) ligand architectures in the complexes improves the biological (anti-proliferative) activity of the complexes by reducing the chance of ligand dissociation under physiological conditions. In general, the pincer (SNS) osmium complexes were found to be more cytotoxic than their ruthenium analogues, suggesting that the anti-proliferative activity of the imidazole-2-thione-Ru/Os complexes depends on the ligand's spatial coordination, the nature of the metal center, and the charge of the metal complex ions.

Phenolic Determination in Proembryogenic Cell Complexes of Buckwheat Morphogenic Cell Culture with Osmium Tetroxide, Toluidine Blue O Dye, and Iron Chloride.

The study of the localization of secondary metabolites in both plants and the cell cultures on the intravital sections is hampered by the difficulty of obtaining thin, correctly oriented sections. Techniques for fixing tissues in resins allow these difficulties to be overcome. Properly selected tissue fixation techniques allow using different dyes to identify the compound of interest. In addition, some components of tissue fixation can act as fixatives and as a dye for identifying secondary metabolites. For example, osmium tetroxide, which fixes lipids in tissues, stains phenolic compounds black. This paper describes methods for the detection of phenolic compounds in morphogenic callus culture of buckwheat using osmium tetroxide, Toluidine Blue O dye, and ferric chloride as dyes in epoxy resin-embedded cell culture with double fixation of the material and when material fixed in Karnovsky's fixative.

Glucose Oxidase Energized Osmium with Dual-Active Centers and Triple Enzyme Activities for Infected Diabetic Wound Management.

Diabetic wounds are susceptible to bacterial infections, largely linked to high blood glucose levels (hyperglycemia). To treat such wounds, enzymes like glucose oxidase (GOx) can be combined with nanozymes (nanomaterials mimic enzymes) to use glucose effectively for purposes. However, there is still room for improvement in these systems, particularly in terms of process simplification, enzyme activity regulation, and treatment effects. Herein, the approach utilizes GOx to directly facilitate the biomineralized growth of osmium (Os) nanozyme (GOx-OsNCs), leading to dual-active centers and remarkable triple enzyme activities. Initially, GOx-OsNCs use vicinal dual-active centers, enabling a self-cascaded mechanism that significantly enhances glucose sensing performance compared to step-by-step reactions, surpassing the capabilities of other metal sources such as gold and platinum. In addition, GOx-OsNCs are integrated into a glucose-sensing gel, enabling instantaneous visual feedback. In the treatment of infected diabetic wounds, GOx-OsNCs exhibit multifaceted benefits by lowering blood glucose levels and exhibiting antibacterial properties through the generation of hydroxyl free radicals, thereby expediting healing by fostering a favorable microenvironment. Furthermore, the catalase-like activity of GOx-OsNCs aids in reducing oxidative stress, inflammation, and hypoxia, culminating in improved healing outcomes. Overall, this synergistic enzyme-nanozyme blend is user-friendly and holds considerable promise for diverse applications.

Photocatalytic CO2 Reduction Using an Osmium Complex as a Panchromatic Self-Photosensitized Catalyst: Utilization of Blue, Green, and Red Light.

The photocatalytic reduction of carbon dioxide (CO2) represents an attractive approach for solar-energy storage and leads to the production of renewable fuels and valuable chemicals. Although some osmium (Os) photosensitizers absorb long wavelengths in the visible-light region, a self-photosensitized, mononuclear Os catalyst for red-light-driven CO2 reduction has not yet been exploited. Here, we discovered that the introduction of an Os metal to a PNNP-type tetradentate ligand resulted in the absorption of light with longer-wavelength (350-700 nm) and that can be applied to a panchromatic self-photosensitized catalyst for CO2 reduction to give mainly carbon monoxide (CO) with a total turnover number (TON) of 625 under photoirradiation (λ≥400 nm). CO2 photoreduction also proceeded under irradiation with blue (λ0=405 nm), green (λ0=525 nm), or red (λ0=630 nm) light to give CO with >90 % selectivity. The quantum efficiency using red light was determined to be 12 % for the generation of CO. A catalytic mechanism is proposed based on the detection of intermediates using various spectroscopic techniques, including transient absorption, electron paramagnetic resonance, and UV/Vis spectroscopy.

Three-dimensional analysis of the intracellular architecture by scanning electron microscopy.

The two-dimensional observation of ultrathin sections from resin-embedded specimens provides insufficient understanding of the three-dimensional (3D) morphological information of membranous organelles. The osmium maceration method, developed by Professor Tanaka's group over 40 years ago, is the only technique that allows direct observation of the 3D ultrastructure of membrane systems using scanning electron microscopy (SEM), without the need for any reconstruction process. With this method, the soluble cytoplasmic proteins are removed from the freeze-cracked surface of cells while preserving the integrity of membranous organelles, achieved by immersing tissues in a diluted osmium solution for several days. By employing the maceration method, researchers using SEM have revealed the 3D ultrastructure of organelles such as the Golgi apparatus, mitochondria, and endoplasmic reticulum in various cell types. Recently, we have developed new SEM techniques based on the maceration method to explore further possibilities for this method. These include: (1) a rapid osmium maceration method that reduces the reaction duration of the procedure, (2) a combination method that combines agarose embedding with osmium maceration to elucidate the 3D ultrastructure of organelles in free and cultured cells, and (3) a correlative immunofluorescence and SEM technique that combines cryosectioning with the osmium maceration method, enabling the correlation of the immunocytochemical localization of molecules with the 3D ultrastructure of organelles. In this paper, we review the novel osmium maceration methods described above and discuss their potential and future directions in the field of biology and biomedical research.

Dermal discoloration due to osmium tetroxide.

INTRODUCTION: Osmium tetroxide is a strong oxidizing agent. After dermal exposure to osmium tetroxide, skin discoloration and red papules can occur. We describe a patient with skin discoloration due to osmium tetroxide. CASE SUMMARY: A 25-year-old postgraduate student unintentionally exposed his hand to osmium tetroxide while working in a laboratory setting. After immediate washing, he sought medical care due to left middle finger discoloration. He reported no discomfort in the affected area. Thorough water rinsing was continued, and corticosteroid ointment was applied. IMAGES: Our patient developed dark brown pigmentation on the ventral side of the left middle finger. The pigmentation disappeared one week later. CONCLUSION: Osmium tetroxide may induce dark brown skin discoloration.

(n,2n) cross section calculations for tungsten, tantalum and osmium nuclei.

Tungsten, tantalum and osmium are important alloying elements in the nuclear technology research and development, particularly in nuclear fission/fusion power plant material applications. So, data results of the cross sections and emission spectra of neutron-induced reactions are required to predict nuclear responses in these elements. However, the cross sections measurements of (n,2n) reactions on tungsten, tantalum and osmium isotopes are rather limited in the literature. In this case, theoretical approaches are often used for obtaining the cross section data. In this article, theoretical (n,2n) cross sections on 180,182-184,186W, 181Ta and 186,192Os target nuclei are calculated up to 20 MeV energy, using the simulation codes TALYS 1.95, ALICE/ASH and CEM03.01. Further, the empirical (n,2n) systematics based on the statistical model have been used for predicting the cross section data at ∼14 MeV incident neutrons. The present results from the empirical systematics and model-based calculations are also compared with the literature experimental data, and JENDL-5.0, ENDF/B-VIII, JEFF3.3 and TENDL-2021 libraries. This paper can provide a contribution to complete description of the (n,2n) reactions considering the lack of experimental cross section data.

Virus-templated redox nanowire network for enzyme electrode.

Viruses have unique coat proteins that are genetically modifiable. Their surface can serve as a nano-template on which electroactive molecules are immobilized. In this study, we report filamentous bacteriophage as a backbone to which redox mediators are covalently and densely tethered, constructing redox nanowire, i.e. an electron conducting biomaterial. The highly ordered coat proteins of a filamentous bacteriophage provide flexible and biocompatible platform to constitute a biohybrid redox nanowire. Incorporating bacteriophage and redox molecules form an entangled assembly of nanowires enabling facile electron transfer. Electron transfer among the molecular mediators in the entangled assembly originates apparent electron diffusion of which the electron transfer rate is comparable to that observed in conventional redox polymers. Programming peptide terminals suggests further enhancement in electron mediation by increasing redox species mobility. In addition, the redox nanowire film functions as a favorable matrix for enzyme encapsulation. The stability of the enzymes entrapped in this unique matrix is substantially improved.

A conventional fixation volume electron microscopy protocol for plants.

Volume electron microscopy techniques play an important role in plant research from understanding organelles and unicellular forms to developmental studies, environmental effects and microbial interactions with large plant structures, to name a few. Due to large air voids central vacuole, cell wall and waxy cuticle, many plant tissues pose challenges when trying to achieve high quality morphology, metal staining and adequate conductivity for high-resolution volume EM studies. Here, we applied a robust conventional chemical fixation strategy to address the special challenges of plant samples and suitable for, but not limited to, serial block-face and focused ion beam scanning electron microscopy. The chemistry of this protocol was modified from an approach developed for improved and uniform staining of large brain volumes. Briefly, primary fixation was in paraformaldehyde and glutaraldehyde with malachite green followed by secondary fixation with osmium tetroxide, potassium ferrocyanide, thiocarbohydrazide, osmium tetroxide and finally uranyl acetate and lead aspartate staining. Samples were then dehydrated in acetone with a propylene oxide transition and embedded in a hard formulation Quetol 651 resin. The samples were trimmed and mounted with silver epoxy, metal coated and imaged via serial block-face scanning electron microscopy and focal charge compensation for charge suppression. High-contrast plant tobacco and duckweed leaf cellular structures were readily visible including mitochondria, Golgi, endoplasmic reticulum and nuclear envelope membranes, as well as prominent chloroplast thylakoid membranes and individual lamella in grana stacks. This sample preparation protocol serves as a reliable starting point for routine plant volume electron microscopy.

Application of immuno- and affinity labeling with fluorescent dyes to in-resin CLEM of Epon-embedded cells.

In-resin CLEM (Correlative Light and Electron Microscopy) of Epon-embedded cells involves correlating fluorescence microscopy with electron microscopy in the same Epon-embedded ultrathin section. This method offers the advantage of high positional accuracy compared to standard CLEM. However, it requires the expression of recombinant proteins. In order to detect the localization of endogenous target(s) and their localized ultrastructures of Epon-embedded samples using in-resin CLEM, we investigated whether immunological and affinity-labeling using fluorescent dyes applied to in-resin CLEM of Epon-embedded cells. The orange fluorescent (λem ∼550 nm) and far-red (λem ∼650 nm) fluorescent dyes examined maintained a sufficient level of fluorescent intensity after staining with osmium tetroxide and subsequent dehydration treatment with ethanol. Immunological in-resin CLEM of mitochondria and the Golgi apparatus was achieved using anti-TOM20, anti-GM130 antibodies, and fluorescent dyes. Two-color in-resin CLEM revealed that wheat germ agglutinin-puncta showed the ultrastructures of multivesicular body-like structures. Finally, taking the advantage of high positional accuracy, volume in-resin CLEM of mitochondria in the semi-thin section (2 µm thick) of Epon-embedded cells was performed by focused ion beam scanning electron microscopy. These results suggested that the application of immunological reaction and affinity-labeling with fluorescent dyes to in-resin CLEM of Epon-embedded cells is suitable for analyzing the localization of endogenous targets and their ultrastructures by scanning and transmission electron microscopy.

Exploration of Optimal pH in Hypothermic Machine Perfusion for Rat Liver Grafts Retrieved after Circulatory Death.

Ex vivo hypothermic machine perfusion (HMP) is a strategy for controlling ischemia-reperfusion injury in donation after circulatory death (DCD) liver transplantation. The pH of blood increases with a decrease in temperature and water dissociation, leading to a decrease in [H+]. This study aimed to verify the optimal pH of HMP for DCD livers. Rat livers were retrieved 30 min post-cardiac arrest and subjected to 3-h cold storage (CS) in UW solution (CS group) or HMP with UW-gluconate solution (machine perfusion [MP] group) of pH 7.4 (original), 7.6, 7.8, and 8.0 (MP-pH 7.6, 7.8, 8.0 groups, respectively) at 7-10 °C. The livers were subjected to normothermic perfusion to simulate reperfusion after HMP. All HMP groups showed greater graft protection compared to the CS group due to the lower levels of liver enzymes in the former. The MP-pH 7.8 group showed significant protection, evidenced by bile production, diminished tissue injury, and reduced flavin mononucleotide leakage, and further analysis by scanning electron microscopy revealed a well-preserved structure of the mitochondrial cristae. Therefore, the optimum pH of 7.8 enhanced the protective effect of HMP by preserving the structure and function of the mitochondria, leading to reduced reperfusion injury in the DCD liver.

Electrochemical and biosensing properties of an FAD-dependent glucose dehydrogenase from Trichoderma virens.

We investigated the bioelectrochemical properties of an FAD-dependent glucose dehydrogenase from Trichoderma virens (TvGDH) and its electrochemical behaviour when immobilized on a graphite electrode. TvGDH was recently shown to have an unusual substrate spectrum and to prefer maltose over glucose as substrate, and hence could be of interest as recognition element in a maltose sensor. In this study, we determined the redox potential of TvGDH, which is -0.268 ± 0.007 V vs. SHE, and advantageously low to be used with many redox mediators or redox polymers. The enzyme was entrapped in, and wired by an osmium redox polymer (poly(1-vinylimidazole-co-allylamine)-{[Os(2,2'-bipyridine)2Cl]Cl}) with formal redox potential of +0.275 V vs. Ag|AgCl via poly(ethylene glycol) diglycidyl ether crosslinking onto a graphite electrode. When the TvGDH-based biosensor was tested with maltose it showed a sensitivity of 1.7 µA mM-1cm-2, a linear range of 0.5-15 mM, and a detection limit of 0.45 mM. Furthermore, it gave the lowest apparent Michaelis-Menten constant (KM app) of 19.2 ± 1.5 mM towards maltose when compared to other sugars. The biosensor is also able to detect other saccharides including glucose, maltotriose and galactose, these however also interfere with maltose sensing.

Cyclization, Decyclization, and Metallacyclization of Pyridine-Substituted Homopropargylic Alcohols on Ruthenium(II) and Osmium(II) Centers.

Systematic investigations on the reactions between cis-[M(dppm)2 Cl2 ] (M=Ru/Os; dppm=1,1-bis(diphenylphosphino)methane) and pyridine/quinoline substituted homopropargylic alcohols uncovered the diverse Ru(II)/Os(II)-induced alkyne activation pathways. The alkynes underwent cyclization on M via a "non-vinylidene" pathway at lower temperatures, resulting in alkenyl intermediates which might further metallacyclize to give metallapyrroloindolizines. Conversely, reactions at higher temperatures induced alkyne cyclization on M via a "vinylidene" pathway, affording cyclic oxacarbene complexes. Additionally, a rare decyclization mechanism was observed during the transformation of a metallacyclization-resistant alkenyl complex into a cyclic oxacarbene complex. DFT calculations were employed to validate the experimental findings. Overall, these results not only provide insights into controlling alkyne activation pathways, but also offer new strategies for preparing metalated heterocyclic and metallacyclic complexes.