Gold compounds for catalysis and metal-mediated transformations in biological systems

One of the challenges of modern inorganic chemistry is translating the potential of metal catalysts to living systems to achieve controlled non-natural transformations. This field poses numerous issues associated with the metal compounds biocompatibility, stability, and reactivity in complex aqueous environment. Moreover, it should be noted that although referring to ‘metal catalysis’, turnover has not yet been fully demonstrated in most of the examples within living systems. Nevertheless, transition metal catalysts offer an opportunity of modulating bioprocesses through reactions that are complementary to enzymes. In this context, gold complexes, both coordination and organometallic, have emerged as promising tools for bio-orthogonal transformations, endowed with excellent reactivity and selectivity, compatibility within aqueous reaction medium, fast kinetics of ligand exchange reactions, and mild reaction conditions. Thus, a number of examples of goldtemplated reactions in a biologically relevant context will be presented and discussed here in relation to their potential applications in biological and medicinal chemistry. Addresses 1 School of Chemistry, Cardiff University, Main Building, Park Place, CF10 3AT, Cardiff, United Kingdom 2 Chair of Medicinal and Bioinorganic Chemistry, Department of Chemistry, Technical University of Munich, Lichtenbergstr. 4, 85748, Garching, Germany Corresponding author: Casini, Angela (angela.casini@tum.de) Current Opinion in Chemical Biology 2020, 55:103–110 This reviews comes from a themed issue on Bioinorganic chemistry Edited by Victoria J. DeRose and Sarah Michel For a complete overview see the Issue and the Editorial https://doi.org/10.1016/j.cbpa.2019.12.007 1367-5931/© 2020 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.o rg/licenses/by/4.0/).


Introduction
The use of metal complex catalysts within living biological systems is an area of research that has recently gained significant attention [1e6]. Metal-mediated reactions have potential use for biological sensing, imaging, and caging applications, as well as for therapy. Certainly, the peculiar reactivity and selectivity of metal-based complexes significantly broadens the scope of the chemical reaction toolbox for biomolecule modification. Current examples of metal-catalysed reactions in biological settings and of therapeutic relevance include redox-active transition metal complexes that can be used to promote the formation of reactive oxygen species or metal-bound oxygen species, responsible for subsequent oxidative damage to biological targets [7e 9], and that are regenerated through electron transfer from intracellular reducing biomolecules [10]. In this category of reactive oxygen speciesegenerating compounds, metal complexes producing singlet oxygen inside cells upon irradiation with light (photodynamic therapy, PDT) are relevant, with some examples already close to clinical application [11].
In addition to redox processes, the chemistry of transition metal complexes has been rapidly expanding to other types of transformations in living conditions, including cross-coupling reactions [12e14], cycloadditions [15], hydrogenation and transfer hydrogenation reactions [16,17], or functional group deprotection (uncaging) reactions [18e21]. It is worth mentioning that the past decade witnessed new ways of tagging proteins with fluorophores or other probes based on metal-templated mechanisms including the Suzukie Miyaura, MizorokieHeck and Sonogashira crosscoupling reactions [4,5,22]. In this context, palladium compounds occupy a pivotal role [5]. Although the field is dominated by homogenous metal-based compounds, heterogeneous Pd catalysts hold promise [23,24], with potential for in vivo application. Recent work in the metal promoted CeH functionalization of nucleobases in aqueous media might also lead to future biological applications [25]. All these processes can be exploited for therapy, both to deactivate relevant pharmacological targets and to induce intracellular redox damage, or for prodrug activation in situ, as well as for bio-orthogonal modifications of biomolecules in physiological environment enabling studies of biochemical interactions on the single-molecule level.
biomolecules via CeC or C-X (X = heteroatom) bond formation for different applications in biological systems. This is not surprising because the power of gold catalysis also stems from the ability of gold cationic complexes to coordinate and activate unsaturated bonds in a chemoselective manner [26e28]. In this review, we will critically present various examples of goldtemplated reactions, illustrating the compounds' design concept and proposed mechanisms, as well as the advantages with respect to other metal-mediated approaches. Reports on the selective sensing of 'free' gold ions in cells will be included, in which the ability of Au III /Au I ions to promote cyclization and fluorescence activation of organic molecules containing alkyne groups is exploited. The use of gold-mediated cross-coupling reactions for selective modification of proteins will also be discussed. Furthermore, examples of catalytic gold complexes and their potential therapeutic applications will be highlighted.

Gold compounds for metal-mediated bioorthogonal modifications and catalysis in cells
To the best of our knowledge, the first report of gold compounds as catalysts in biological conditions was published by Jou et al. in 2009, [29**] when a rhodamineealkyne derivative was investigated as a selective fluorescence sensor d 'chemodosimeter' d for Au III ions. In detail, the Au III ions mediated the intramolecular cyclization of the propargylamide moiety on the rhodamine derivative to an oxazolecarbaldehyde moiety ( Figure 1, Table 1). This instigated over 100-fold enhancement in the substrate fluorescence and induced a colorimetric change to pink from colourless [29**]. This effect was also previously observed in the presence of Pd 2þ salts; however, addition of stoichiometric amount of an oxidant was required in this latter case [30]. The selectivity of the sensor was evaluated in the presence of a variety of metal ions, including divalent cations of the transition metal series and alkaline and alkaline earth ions; however, fluorescence was produced only in the presence of Au III ions, illustrating its exceptional selectivity [29**]. To elucidate the applicability of the rhodamineealkyne probe in living systems, the reaction was investigated by fluorescence live cell imaging using HaCaTcells (human keratinocyte cell line). The cells were first incubated with the non-fluorescent probe (20 mM) for 2 h and then with AuCl 3 (10 mM) for 1 h. Confocal microscopy images showed intracellular localization of the fluorescence, demonstrating the potential of the probe for applications in biological systems [29**]. In the same year, Yang et al. [31] published a similar work describing the use of a slightly different rhodamine derivative that could act as a probe for Au III ions ( Table 1). As in the previous example, the irreversible Au III -promoted cyclization of the nonfluorescent rhodamine amide tethered with an alkyne to a highly fluorescent compound was observed by fluorescence spectroscopy and microscopy in cells [31].
An alternative Au III ion sensor was published one year later by Do et al. [34**] based on an apo-coumarin scaffold rather than the propargylamide-derived rhodamine previously reported [[29**], [31]]. The latent apo-coumarin fluorophore contained a dialkylamino group in para position to the Michael acceptor, favouring the Au III -mediated hydroarylation [34**]. The cyclization reaction via CeC bond formation led to a marked increase in fluorescence which could be observed also in HaCaT cells (Table 1) (Table 1). In this case, coordination complexes of the type [Au I (L)Cl] (L = phosphane ligand) were used. Thus, the complex [Au(PTA)Cl] (PTA = 1,3,5-triaza-7phosphaadamantane) (5 mol%) was tested and the reaction, performed in water/acetonitrile (20% vol) at 37 C, resulted in excellent yields [41**]. Afterwards,  Unfortunately, the presence of excess thiols such as glutathione and cysteine, typically found in physiological conditions, led to a marked inhibition of the catalytic ability of [Au(PTA)Cl], most likely due to the competitive binding of the thiols to the gold [41**]. Therefore, these types of complexes may have scarce chances to be catalytically active in vivo unless properly targeted to cancer tissues and protected from speciation. Of note, this work also included a concurrent Rumediated deallylation reaction alongside the Aumediated hydroarylation, with the same substrates [41**]. Infrared fluorescence was observed for the deallylation product, whereas the hydroarylation product produced green and blue emission as seen previously. This is the first example of concurrent and orthogonal Au/Ru-mediated intracellular reactions, providing an initial step towards the production of artificial metabolic networks mediated by metals [41**].  Figure 2). This approach was proposed as an alternative to the prevalent N-methylmaleimide cysteine ligation, which suffered with lack of stability in physiological conditions [22]. In detail, the modular synthesis of four cyclometalated Au III C^N complexes was reported, including [Au(C CH2 N)msen] (C CH2 N = 2-benzylpyridine; msen = N,N 0 -  The biocompatibility of the organometallic Au III system was demonstrated further by cysteine arylation of designed ankyrin repeat protein and fibroblast growth factor 2, as observed by LC-MS analysis [36**]. Afterwards, the Au III compound was challenged against a comparable Pd II complex reported for cysteine arylation [42], with respect to GSH arylation [36**]. The results showed ca. 92% conversion to the Au-mediated conjugate, indicating that substrate arylation occurred at a quicker rate than the palladium-mediated arylation [36**]. The success of gold-mediated cysteine arylation by [Au III (C CH2 N)Cl 2 ] (1, Figure 2) was confirmed by de Paiva et al. [37*], observing the reaction in zinc finger (ZF) protein domains by MS. Of note, in this case, reductive elimination was observed only in reaction conditions far from the physiological ones and after 48 h incubation [37*]. Moreover, classical Au III complexes with bidentate N-donor ligands were ineffective with respect to Cys arylation, although able to form Au-ZF adducts.
Inspired by these results, our group recently expanded the library of Au III cyclometalated (C^N) complexes capable of cysteine arylation, including 1, complex Au(C NH N)Cl 2 (2, C NH N = N-phenylpyridin-2-amine) and Au(C CO N)Cl 2 (3, C CO N = 2-benzoylpyridine) ( Figure 2)  To elucidate the mechanism leading to reductive elimination and to draw initial structure activity relationships, DFT (density functional theory) calculations were performed on the reaction of the complexes with two cysteinate ligands used as models [38**]. Thus, a reaction mechanism was proposed, whereby the first cysteinate binds trans to the N and the second one is needed to favour the bond breakage between the nitrogen and the Au III centre forming the intermediate [Au III (C^N)(Cys) 2 Cl] -. This enables rotation of the aryl group, allowing reductive elimination to take place forming the Cys-arylated product and the [CysAu I Cl]side complex. The calculated relative standard free Gibbs energy values provided a possible explanation for the increased ability of Au(C CO N)Cl 2 to perform reductive elimination compared with Au(C CH2 N)Cl 2 and even more so Au(C NH N)Cl 2 . In detail, the activation barrier for the CeS coupling increases in the order 3 < 1 < 2, following the same trend as the experimental reductive elimination reaction rate [38**].
In 2017, the first in vivo study on a gold-templated reaction was published by Tsubokura et al. [39**], whereby a Au III cyclometalated compound conjugated to coumarin was capable of activating propargyl esters featuring another fluorophore (Table 1, Figure 3). In this case, Au III -mediated amide bond formation occurs between the propargyl ester probes and nearby proteinsurface amines. The work exploited the use of organtargeting glycoalbumins which acted as carriers for the Au-coumarin catalyst owing to the strong binding affinity between the hydrophobic coumarin and the binding pocket of albumin [[39**], [46]]. The compound enabled localization in specific organs in mice by fluorescence imaging (Figure 3), providing the proof of concept for a more therapeutically viable Au catalyst [39**].

Summary and perspectives
The use of transition metal catalysis within living systems is nontrivial, due to stability, efficiency and potential poisoning of the catalysts. Nevertheless, this field has gained importance over the past few years and provided a number of promising examples. In this context, gold-promoted reactions represent a significant addition to the toolbox of life compatible transformations. As discussed here, recent reports explore gold compounds for a number of metal-mediated bioorthogonal transformations in living systems, specifically aiming at producing novel chemical tools and therapeutic agents. Although until now the few available examples suffer from the side reactivity of the Au I /Au III complexes with biomolecules and intracellular reducing agents which prevent their catalytic action, in the near future this could be overcome by fine-tuning the compounds' redox and nucleophilic properties via judicious choice of the ligand system. For example, different families of organometallic gold complexes, endowed with improved stability and robustness of functionalization, could be explored, including Au I N-heterocyclic carbenes and alkynyl complexes, as well as cyclometalated Au III compounds [47,48]. This strategy has already been exploited in classical medicinal inorganic chemistry, where organometallic gold compounds have shown promising anticancer properties in vitro and in vivo [48]. Furthermore, the incorporation of the gold catalysts into engineered nanometric scaffold (to achieve socalled 'nanozymes') could be envisaged, with the aim of enhancing their water solubility and to provide protection from speciation in vivo [49*].

Conflict of interest statement
Nothing declared.