Exporting Metal-Carbene Chemistry to Live Mammalian Cells: Copper-Catalyzed Intracellular Synthesis of Quinoxalines Enabled by N-H Carbene Insertions

Implementing catalytic organometallic transformations in living settings can offer unprecedented opportunities in chemical biology and medicine. Unfortunately, the number of biocompatible reactions so far discovered is very limited, and essentially restricted to uncaging processes. Here, we demonstrate the viability of performing metal carbene transfer reactions in live mammalian cells. In particular, we show that copper (II) catalysts can promote the intracellular annulation of alpha-keto diazocarbenes with ortho-amino arylamines, in a process that is initiated by an N-H carbene insertion. The potential of this transformation is underscored by the in cellulo synthesis of a product that alters mitochondrial functions, and by demonstrating cell selective biological responses using targeted copper catalysts. Considering the wide reactivity spectrum of metal carbenes, this work opens the door to significantly expanding the repertoire of life-compatible abiotic reactionsThis work has received financial support from Spanish grants (PID2019-108624RB-I00, RTI2018-093813-J-I00 and ORFEO-CINQA network CTQ2016-81797-REDC), the Consellería de Cultura, Educación e Ordenación Universitaria (2015-CP082, ED431C-2017/19 and Centro Singular de Investigación de Galicia accreditation 2019–2022, ED431G 2019/03), the European Union (European Regional Development Fund-ERDF corresponding to the multiannual financial framework 2014–2020), and the European Research Council (Advanced Grant No. 340055). S.G. thanks the European Union (European Regional Development Fund, Interreg V-A POCTEP España-Portugal programme, 2iqbioneuro project) and M.T.G. thanks the financial support from the Agencia Estatal de Investigación (RTI2018-093813-J-I00)S

Intracellular Ruthenium‐Promoted (2+2+2) Cycloadditions

This is the peer reviewed version of the following article: J. Miguel-Ávila, M. Tomás-Gamasa, J. L. Mascareñas, Angew. Chem. Int. Ed. 2020, 59, 17628, which has been published in final form at https://doi.org/10.1002/anie.202006689. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived VersionsMetal‐mediated intracellular reactions are becoming invaluable tools in chemical and cell biology, and hold promise for strongly impacting the field of biomedicine. Most of the reactions reported so far involve either uncaging or redox processes. Demonstrated here for the first time is the viability of performing multicomponent alkyne cycloaromatizations inside live mammalian cells using ruthenium catalysts. Both fully intramolecular and intermolecular cycloadditions of diynes with alkynes are feasible, the latter providing an intracellular synthesis of appealing anthraquinones. The power of the approach is further demonstrated by generating anthraquinone AIEgens (AIE=aggregation induced emission) that otherwise do not go inside cells, and by modifying the intracellular distribution of the products by simply varying the type of ruthenium complexThis work has received financial support from the Spanish Government (SAF2016‐76689‐R, ORFEO‐CINQA network CTQ2016‐81797‐REDC) the Consellería de Cultura, Educación e Ordenación Universitaria (2015‐CP082, ED431C‐2017/19 and Centro Singular de Investigación de Galicia Accreditation 2019–2022, ED431G 2019/03), the European Union (European Regional Development Fund‐ERDF corresponding to the multiannual financial framework 2014–2020), and the European Research Council (Advanced Grant No. 340055). J.M.Á. thanks the Ministerio de Educación, Cultura y Deporte for the FPU fellowship (FPU16/00711) and M.T.G. thanks the financial support from the Agencia Estatal de Investigación (RTI2018‐093813‐J‐I00)S

Transition metal catalysis in the mitochondria of living cells

The development of transition metal catalysts capable of promoting non-natural transformations within living cells can open significant new avenues in chemical and cell biology. Unfortunately, the complexity of the cell makes it extremely difficult to translate standard organometallic chemistry to living environments. Therefore, progress in this field has been very slow, and many challenges, including the possibility of localizing active metal catalysts into specific subcellular sites or organelles, remain to be addressed. Herein, we report a designed ruthenium complex that accumulates preferentially inside the mitochondria of mammalian cells, while keeping its ability to react with exogenous substrates in a bioorthogonal way. Importantly, we show that the subcellular catalytic activity can be used for the confined release of fluorophores, and even allows selective functional alterations in the mitochondria by the localized transformation of inert precursors into uncouplers of the membrane potentialWe are thankful for the support given by the Spanish grant SAF2013-41943-R, the Xunta de Galicia (GRC2013-041 and 2015-CP082), the ERDF and the European Research Council (Advanced Grant No. 340055). M.T.G. thanks the Ministerio de Economía y Competitividad for the Postdoctoral fellowship. M.M.C. thanks Ministerio de Economía y Competitividad for the Juan de la Cierva-Incorporacio´n fellowship (IJCI-2014-19326).S

Ruthenium-Catalyzed Redox Isomerizations inside Living Cells

Tailored ruthenium(IV) complexes can catalyze the isomerization of allylic alcohols into saturated carbonyl derivatives under physiologically relevant conditions, and even inside living mammalian cells. The reaction, which involves ruthenium-hydride intermediates, is bioorthogonal and biocompatible, and can be used for the “in cellulo” generation of fluorescent and bioactive probes. Overall, our research reveals a novel metal-based tool for cellular intervention, and comes to further demonstrate the compatibility of organometallic mechanisms with the complex environment of cellsThis work has received financial support from the Spanish Government (SAF2016-76689-R, Orfeo-cinqa network CTQ2016-81797-REDC), the Consellería de Cultura, Educación e Ordenación Universitaria (2015-CP082, ED431C 2017/19), Centro Singular de Investigación de Galicia accreditation 2016–2019, ED431G/09), the European Union (European Regional Development Fund-ERDF), and the European Research Council (Advanced Grant No. 340055)S

Plasmonic-assisted thermocyclizations in living cells using metal−organic framework based nanoreactors

We describe a microporous plasmonic nanoreactor to carry out designed near-infrared (NIR)-driven photothermal cyclizations inside living cells. As a proof of concept, we chose an intramolecular cyclization that is based on the nucleophilic attack of a pyridine onto an electrophilic carbon, a process that requires high activation energies and is typically achieved in bulk solution by heating at ∼90 °C. The core–shell nanoreactor (NR) has been designed to include a gold nanostar core, which is embedded within a metal–organic framework (MOF) based on a polymer-stabilized zeolitic imidazole framework-8 (ZIF-8). Once accumulated inside living cells, the MOF-based cloak of NRs allows an efficient diffusion of reactants into the plasmonic chamber, where they undergo the transformation upon near-IR illumination. The photothermal-driven reaction enables the intracellular generation of cyclic fluorescent products that can be tracked using fluorescence microscopy. The strategy may find different type of applications, such as for the spatio-temporal activation of prodrugsThe authors thank the financial support of the MCIN/AEI (PID2020-119206RB-I00, PID2019-108624RB-I00, CTQ2017-84767-P, RYC-2017-23457, RYC-2019-028238-I, RTI2018-093813-J-I00), the Xunta de Galicia (ED431F 2017/02, 2021-CP054, ED431C-2021/25, Centro Singular de Investigación de Galicia Accreditation 2019−2022, and ED431G 2019/03), the European Union (European Regional Development Fund − ERDF; H2020-MSCA-IF grant agreement no. 749667; H2020-MSCA-ITN grant agreement no. 860942; H2020-FET-Open grant agreement No. 899612; and INTERREG V-A Spain−Portugal, project 0624_2IQBIONEURO_6_E), and the European Research Council (starting grant no. 950421, advanced grant no. 340055). The support of the orfeo-cinqa network (CTQ2016-81797-REDC) is also kindly acknowledgedS

Core-Shell Palladium/MOF Platforms as Diffusion-Controlled Nanoreactors in Living Cells and Tissue Models

Translating the potential of transition metal catalysis to biological and living environments promises to have a profound impact in chemical biology and biomedicine. A major challenge in the field is the creation of metal-based catalysts that remain active over time. Here, we demonstrate that embedding a reactive metallic core within a microporous metal-organic framework-based cloak preserves the catalytic site from passivation and deactivation, while allowing a suitable diffusion of the reactants. Specifically, we report the fabrication of nanoreactors composed of a palladium nanocube core and a nanometric imidazolate framework, which behave as robust, long-lasting nanoreactors capable of removing propargylic groups from phenol-derived pro-fluorophores in biological milieu and inside living cells. These heterogeneous catalysts can be reused within the same cells, promoting the chemical transformation of recurrent batches of reactants. We also report the assembly of tissue-like 3D spheroids containing the nanoreactors and demonstrate that they can perform the reactions in a repeated mannerThe authors thank the financial support of the MINECO ( CTQ2017-89588-R , SAF2016-76689-R , CTQ2017-84767-P , RYC-2014-16962 , and RYC-2017-23457 ), the Xunta de Galicia ( ED431F 2017/02 , 2015-CP082 , ED431C 2017/19 , and Centro singular de investigación de Galicia accreditation 2019-2022, ED431G 2019/03 ), the European Union (European Regional Development Fund [ERDF]; H2020-MSCA-IF-2016 grant agreement no. 749667 ; and INTERREG V-A Spain-Portugal [POCTEP] 2014-2020, project 0624_2IQBIONEURO_6_E ), and the European Research Council (advanced grant no. 340055 ). Support of the orfeo-cinqa network ( CTQ2016-81797-REDC ) is also kindly acknowledgedS

Ruthenium-Catalyzed Azide–Thioalkyne Cycloadditions in Aqueous Media: A Mild, Orthogonal, and Biocompatible Chemical Ligation

The development of efficient metal-promoted bioorthogonal ligations remains as a major scientific challenge. Demonstrated herein is that azides undergo efficient and regioselective room-temperature annulations with thioalkynes in aqueous milieu when treated with catalytic amounts of a suitable ruthenium complex. The reaction is compatible with different biomolecules, and can be carried out in complex aqueous mixtures such as phosphate buffered saline, cell lysates, fetal bovine serum, and even living bacteria (E. coli). Importantly, the reaction is mutually compatible with the classical CuAACThis work has received financial support from Spanish grants (SAF2016-76689-R and SAF2013-41943-R), the Xunta de Galicia (2015-CP082 and Centro Singular de Investigaciln de Galicia accreditation 2016-2019 ED431G/09), the European Union (European Regional Development Fund - ERDF), and the ERC (Adv. Grant 340055)S

Bright insights into Palladium-triggered local chemotherapy

The incorporation of transition metal catalysts to the bioorthogonal toolbox has opened the possibility of producing supra-stoichiometric amounts of xenobiotics in living systems in a non-enzymatic fashion. For medical use, such metals could be embedded in implantable devices (i.e. heterogeneous catalyst) to “synthesize” drugs in desired locations (e.g. in a tumour) with high specificity and for extended periods of time, overcoming the useful life limitations of current local therapy modalities directed to specific organ sites (e.g. brachytherapy, controlled release systems). To translate this approach into a bona fide therapeutic option, it is essential to develop clinically-accessible implantation procedures and to understand and validate the activation process in relevant preclinical models. Herein we report the development of a novel Pd-activatable precursor of the red-fluorescent drug doxorubicin and Pd devices of optimized size and activity. Screening in state-of-the-art cancer models provided fundamental insights into the insertion protocols, safety and stability of the devices and into the prodrug distribution profile before and after activation