Organometallic catalysis in biological media and living settings

NOTICE:Thisisthepeerreviewedversionofthefollowingarticle:MiguelMartínez‐Calvo,José L.Mascareñas(2018).Organometalliccatalysisinbiologicalmediaandlivingsettings. Coord.Chem.Rev.359,57–79[doi:10.1016/j.ccr.2018.01.011]  ©2018.ThismanuscriptversionismadeavailableundertheCC‐BY‐NC‐ND4.0 licensehttp://creativecommons.org/licenses/by‐nc‐nd/4.0/ 1 2Review 4Organometallic catalysis in biological media and living settings 5 6 7Miguel Martínez-Calvo ⇑ , José L. Mascareñas ⇑ 8Centro Singular de Investigación en Química Biolóxica e Materiais Moleculares (CIQUS) and Departamento de Química Orgánica, Universidade de Santiago de Compostela, 915782 Santiago de Compostela, Spain 10 11 12 14 article info 15 Article history: 16 Received 12 October 2017 17 Accepted 7 January 2018 18 Available online xxxx 19 Keywords: 20 Organometallic 21 Bioorthogonal 22 Transition metal catalysis 23 Living cells 24 Artificial metalloenzymes 25 26 abstract 27 Organometallic catalysis has allowed the development of an impressive number of chemical transforma28 tions that could not be achieved using classical methodologies. Most of these reactions have been accom29 plished in organic solvents, and in many cases in the absence of water, and under air-free conditions. The 30 increasing pressure to develop more sustainable transformations has stimulated the discovery of metal31 catalyzed reactions that can take place in water. A particularly attractive extension of this chemistry con32 sists of the use of biological relevant aqueous solvents, as this might set the basis to translate catalytic 33 metal complexes to biological settings. While this research field is in its infancy, along the last ten years 34 there have been an increasing number of reports demonstrating the viability of achieving metal35 promoted transformations in biologically relevant contexts. In this review, that does not intend to be 36 comprehensive, we summarize the most significant advances in the area, and highlight some of the more 37 important difficulties that must be faced when trying to design biocompatible organometallic catalysts, 38 such us stability, cell uptake, bioorthogonality and toxicity. We will manly focus on transition metal sys39 tems which have been showed to keep their activity in complex aqueous buffers and inside living cells. 40 Ó2018 Published by Elsevier B.V. 41 42 43 44 45 Contents 46 1. Introduction . . . ....................................................................................................... 00 47 2. Transition metal catalysis in biologically settings . . . . . . . . . . . ................................................................. 00 48 2.1. Ruthenium . . . . . . . . . . . . .......................................................................................... 00 49 2.2. Palladium. . . . . . . . . . . . . .......................................................................................... 00 50 2.2.1. Protein modification. . . . . . . . ............................................................................... 00 51 2.2.2. Pd-nanostructured materials . ............................................................................... 00 52 2.3. Copper . . . . . . . . . . . . . . . .......................................................................................... 00 53 2.3.1. Discrete copper complexes for achieving CuAACs in cellular settings . . . . . . ......................................... 00 54 2.3.2. Copper nanostructures . . . . . . ............................................................................... 00 55 2.4. Gold . . . . . . . . . . . . . . . . . .......................................................................................... 00 56 2.5. Iridium . . . . . . . . . . . . . . . .......................................................................................... 00 57 2.6. Iron. . . . . . . . . . . . . . . . . . .......................................................................................... 00 https://doi.org/10.1016/j.ccr.2018.01.011 0010-8545/Ó2018 Published by Elsevier B.V. Abbreviations: CuAAC, Copper-Catalyzed Azide–Alkyne Cycloadditions; PhSH, thiophenol; alloc, allylcarbamate group; DAPI, 4 0 ,6-diamidine-2 0 -phenylindole; EtBr, ethidium bromide; TON, turnover number; GSH, glutathione; PBS, phosphate buffered saline; TPP, triphenylphosphonium; IMM, mitochondrial inner membrane; TMRE, tetramethylrhodamine, ethyl ester; ICP-MS, inductively coupled plasma mass spectrometry; RuAtAC, Ruthenium-Catalyzed Azide–Thioalkyne Cycloadditions; GFP, Green Fluorescent Protein; proc, propargylic-carbamate; Neu, neuramic acid; PdNPs, palladium nanoparticles; 5FU, 5-fluoro-1-propargyl-uracil; TFP, ligand tri-2-furylphosphine; HBSS, Hank’s Balanced Salt Solution; PEG, polyethylene glycol; PLGA, poly lactic acid-co-glycolic acid; ROS, reactive oxygen species; THPTA, tris-(hydroxypropyltriazo lylmethyl)amine; BTTAA, bis[(tert-butyltriazoyl)methyl]-[(2-carboxymethyltriazoyl)methyl]-amine; TBTA, tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine; HPG, homopropargylglycine; TCEP, tris(2-carboxyethyl)phosphine hydrochloride; NaAsc, sodium ascorbate; HEK, human embryonic kidney; FITC, Fluorescein isothiocyanate; OVCAR5, human ovarian cancer cells; CuNPs, copper nanoparticles; E–Cu–NPs, embedded copper nanoparticles; Cu-MONPs, Cu-containing organic nanoparticles; FRET, fluorescence resonance energy transfer; AuNPs, gold nanoparticles; TPP, 5,10,15,20-tetraphenyl-21H,23H-porphine; bPPs, bovine pancreatic polypeptides; ee, enantiomeric excess; PIX, porphyrin IX; TOF, turnover frequency; ATHase, artificial transfer hydrogenase; biot–Sav, biotin–streptavidin; NaPi, sodium phosphate solution. ⇑ Corresponding authors. Fax: +34 881814468. E-mail addresses: [email protected] (M. Martínez-Calvo), [email protected] (J.L. Mascareñas). Coordination Chemistry Reviews xxx (2018) xxx–xxx Contents lists available at ScienceDirect Coordination Chemistry Reviews journal homepage: www.elsevier.com/locate/ccr CCR 112651 No. of Pages 23, Model 5G 8 January 2018 Please cite this article in press as: M. Martínez-Calvo, J.L. Mascareñas, Organometallic catalysis in biological media and living settings, Coord. Chem. Rev. (2018), https://doi.org/10.1016/j.ccr.2018.01.011 58 3. Artificial metalloenzymes . . . . . .......................................................................................... 00 59 4. Conclusions and closing remarks . . . . . . . . . . . . . . . . . . ....................................................................... 00 60 Competing financial interests. . . . .......................................................................................... 00 61 Acknowledgments . . . . . . . . . . . .......................................................................................... 00 62 References . .......................................................................................................... 00 63 64 65 1. Introduction 66 Organometallic catalysis in water is itself a quite new field of 67 research which has been mainly developed within the context of 68 ‘‘Green chemistry” [1–6]. In recent years, there have been many 69 reports on metal-catalyzed reactions that can take place in water, 70 including couplings, isomerizations, cyclizations, cycloadditions 71 or hydrolysis processes. Despite this progress, the number of 72 water-compatible organometallic reactions is still very small when 73 compared with transformations achieved in organic solvents [7,8]. 74 Thus, one should expect many new contributions in the coming 75 years. 76 Given that the basic solvent of biological habitats is water, it is 77 not difficult to envision that some of these transformations might 78 be achieved in bio-relevant media. However, the complexity of bio79 logical solvents, owing to the presence of a high concentration of 80 biomolecules such as thiols or amines which can poison the metal 81 and kill the catalytic activity, makes extremely challenging to 82 translate metal-catalyzed reactions to such media. Even more dif83 ficult is the transfer to living cells, as in this case additional issues 84 such as cell uptake and transport, and especially, side biological 85 activities [9–12], need to be taken into account. In addition, transi86 tion metal speciation should be considered, as this could influence 87 the reactivity as well as the toxicity of the metals [13,14]; however, 88 studies in this area, in the context of metal-promoted reactions in 89 cell culture, are yet lacking. Anyhow, in recent years there have 90 been many reports on the use of metal complexes in complex 91 aqueous buffers, and even in vivo settings [15–18]. While 92 organometallic catalysis in biological media is yet an emerging 93 discipline, it seems clear that being able to achieve non-natural 94 catalytic transformations of exogenous substrates in bio-settings 95 might unleash a new world of opportunities for biological and 96 medicinal research. This can be of great relevance for instance for 97 the in situ generation of drugs, the amplification of optical signals 98 for the detection of biomarkers, or the metal-promoted modifica99 tion of biomolecules, among others. 100 Undoubtedly, one of the key discoveries that has had a more 101 significant impact on the development of biocompatible metal102 catalyzed transformations was the report by Sharpless and by Mel103 dal on the famous Copper-Catalyzed Azide–Alkyne Cycloadditions 104 (CuAAC,Scheme 1)[19,20]. This reaction has changed our capabil105 ity to transform and monitor biomolecules, in some cases even in 106 living atmospheres, in the presence of many other native molecu107 lar components. This type of reactions belongs to what Bertozzi 108 coined as Bioorthogonal Chemistry [21,22]. The copper-promoted 109 annulations can be considered as the first metal-catalyzed reac110 tions that could be achieved in complex aqueous media and even 111 in cell culture media for the modification of cell surface sugars 112 [23–25] and proteins [26]. The cytotoxicity of Cu(I) ions, however, 113 has significantly hindered the application of this reaction in the 114 internal space of living cells. 115 After these ground-breaking and inspiring developments in 116 bioorthogonal chemistry, other research groups started to investi117 gate the applicability of other metals to this new field of research. 118 Nevertheless, moving to cells is not trivial, as the living cell is a 119 very complex, compartmentalized and dynamic entity, with a very 120 high concentration of biomolecules, including thiols. Despite this, 121 recent data suggest that certain transition metal derivatives can Scheme 1. Initial mechanism proposed by Sharpless for the CuAAC [19]. 2M. Martínez-Calvo, J.L. Mascareñas / Coordination Chemistry Reviews xxx (2018) xxx–xxx CCR 112651 No. of Pages 23, Model 5G 8 January 2018 Please cite this article in press as: M. Martínez-Calvo, J.L. Mascareñas, Organometallic catalysis in biological media and living settings, Coord. Chem. Rev. (2018), https://doi.org/10.1016/j.ccr.2018.01.011 122 promote intracellular reactions through typical organometallic 123 mechanisms. Here, we have to remark the pioneering work by 124 Streu and Meggers on the development of Ru(II) catalysts compat125 ible with living cells [27], and by Bradley and co-workers [28] and 126 Chen and co-workers [29] on the development of Pd based sys127 tems. In this review, we will summarize some key developments 128 in the field, paying special attention to Ru and Pd compounds; 129 but we will also discuss some achievements with other metals. 130 At the end of the review we will also briefly comment on recent 131 developments on the preparation of artificial metalloenzymes. 132 This field of research is young, but steadily growing, and a num133 ber of reviews dealing with transition metal catalysis in biological 134 settings [30–33], bioorthogonal protein modifications [34] and 135 artificial metalloenzymes [35–40], have been already published. 136 While the above reviews on metal-promoted reactions in bio137 logical media are either classified by reaction types, or centered 138 on biopolymer modifications, or on specific metals, we use an orga139 nization based on the type of metals. This is particularly illustrative 140 because it gives a comparative idea of the transformative possibil141 ities offered by each of them, and their potential for future 142 applications. 143 Finally, it is important to note that while in some sentences we 144 might write ‘‘metal catalysis”, in most of the examples with living 145 systems, turnover has not yet been fully demonstrated. 146 2. Transition metal catalysis in biologically settings 147 2.1. Ruthenium 148 Organometallic ruthenium complexes have been widely consid149 ered in bioinorganic chemistry, especially because of their anti150 cancer potential [41]. Even some of them have shown relevant 151 biological activities owing to their intracellular catalytic activities, 152 for instance by interfering with the balance of GSH and NAD/ 153 NADH, which leads to cell death [42,43]. Ruthenium complexes 154 have been widely used in catalytic organometallic chemistry, and 155 therefore translating some of these reactions to biological settings 156 is highly promising. The use of Ru(II) organometallic complexes as 157 catalytic promoters of exogenous transformations in cellular set158 ting started with a seminal report by Streu and Meggers in 2006, 159 that demonstrated the viability of achieving a metal promoted 160 release allylcarbamate-protecting groups in the interior of HeLa 161 cells (Fig. 1)[27]. For monitoring the reaction, they used as sub162 strate a bis-allylcarbamate caged rhodamine 110 (1) which was 163 virtually non-fluorescent. The uncaging of this molecule gives rise 164 to rhodamine 110 (2) which emits green light upon excitation. The 165 reaction was carried out using [RuCp ⁄ (COD)Cl] (3) as catalyst 166 (Fig. 2). They first carried out an in vitro exploration of the best con167 ditions to achieve a Ru-promoted cleavage of the allylcarbamate 168 group (alloc) of a protected p-methylaniline, under a variety of 169 aqueous buffers, finding that the reaction is better achieved in 170 the presence of nucleophilic thiols. 171 They then moved to living HeLa Cells, observing that when the 172 cells were pre-incubated with 1(100 l M) for 30 min, washed with 173 PBS buffer solution, and then treated with the ruthenium complex 174 3(20 l M) and thiophenol (PhSH) (500 l M), there is an increase in 175 green light emission due to the release of 2(Fig. 1). A few years 176 later, the same authors carried out a similar deprotection reaction 177 using another Ru(II) organometallic precursor [RuCp ⁄ ( g 6 -pyrene)] 178 PF 6 (4,Fig. 2), that can be activated (detachment of the pyrene 179 ligand) by irradiation with light (k= 330 nm). As in the case of 3, 180 the reaction with 4also needed the presence of PhSH as additive 181 [44]. The next contribution in this area was achieved by the group 182 of Mascareñas et al., who demonstrated that is possible to use the Fig. 1. (a) Representation of the uncaging of 1promoted by RuCp * (COD)Cl (3) and (b) imaging of HeLa cells: (i) before the addition of the Ru catalyst to cells preincubated with 1, and PhSH and (ii) 15 min after the addition of ruthenium complex 3. (b) Adapted by permission [27]. Copyright 2006, Wiley-VCH. M. Martínez-Calvo, J.L. Mascareñas / Coordination Chemistry Reviews xxx (2018) xxx–xxx 3 CCR 112651 No. of Pages 23, Model 5G 8 January 2018 Please cite this article in press as: M. Martínez-Calvo, J.L. Mascareñas, Organometallic catalysis in biological media and living settings, Coord. Chem. Rev. (2018), https://doi.org/10.1016/j.ccr.2018.01.011 183 Ru-promoted reaction to activate DNA binders like 4 0 ,6-Diamidine184 2 0 -phenylindole (DAPI), Ethidium bromide (EtBr) and bisbenza185 midines [45], inside living mammalian cells, by removing inacti186 vating alloc protecting groups from their protected precursors 187 (DAPI-alloc (10), bisbenzamidines-alloc (11b) and EtBr-alloc (12) 188 (Fig. 3)[46]. The authors carried out several control experiments 189 that were consistent with the reaction taking place inside mam190 malian cells. These results set the stage for future developments Fig. 2. Structure of ruthenium complexes used as catalysts inside cells. (a) 3[27] and 4[44]; (b) Kitamura’s derived Ru(IV) catalysts 5a–cemployed by Meggers et al. [49]; (c) catalysts 6–8synthetized by Mascareñas et al., in the study of mitochondrial specific catalysis [51] and (d) second generation of quinoline derived Ru(IV) catalyst (9) reported by Meggers et al. [53]. Fig. 3. (a–c) Schematic representation of caged DNA binders; (d) imaging of cells treated with (i) EtBr–alloc (green channel) (12) and (ii) same cells after the addition the ruthenium complex 3and PhSH, where it can be observed how after the removal of the alloc groups, the initial green light turns into the red light emission of EtBr (red channel) [46]. 4M. Martínez-Calvo, J.L. Mascareñas / Coordination Chemistry Reviews xxx (2018) xxx–xxx CCR 112651 No. of Pages 23, Model 5G 8 January 2018 Please cite this article in press as: M. Martínez-Calvo, J.L. Mascareñas, Organometallic catalysis in biological media and living settings, Coord. Chem. Rev. (2018), https://doi.org/10.1016/j.ccr.2018.01.011 191 on metal-promoted activation of DNA-binding compounds in bio192 logical media. 193 The above uncaging strategies employing Ru(II)Cp ⁄ complexes 194 required the use of relatively large amounts of PhSH as an external 195 nucleophile to ensure turnover, a compound that is toxic to the 196 cells. This problem has been recently solved by Volker and Meggers 197 by using ruthenium(IV) catalysts with quinoline ligands that had 198 been previously described by Kitamura et al., for the catalytic 199 dehydrative allylation of alcohols (Fig. 2b)[47,48]. With these 200 organoruthenium complexes (5,Fig. 2), it was possible to use 201 weaker nucleophiles than PhSH such as glutathione (GSH), which 202 is already present in relatively large amounts inside cells. The 203 authors have proposed the catalytic cycle indicated in the Scheme 2 204 for the uncaging of alloc protected amines [49]. 205 These catalysts were more active than ruthenium complex 3, 206 greatly increasing the turnover number (TON) of the reaction, 207 which could be further modulated by changing the substitution 208 of the position 4 of the quinoline ligand. Thus, the presence of a 209 donor atom at this position increased the catalytic performance 210 (5b,5c,Fig. 2). Importantly, this type of catalysts can be used in 211 the presence of living mammalian cells without the need to add 212 PhSH as additive, since glutathione (GSH), which is already present 213 in the cells, acts as nucleophile (NuH) to close the Ru– p –allyl cat214 alytic cycle. 215 Despite all data suggested that the Ru-catalyzed uncaging is 216 highly effective in the presence of living cells, the intracellular nat217 ure of the process was not unambiguously demonstrated. 218 Noticeable, a paper published by Waymouth and Wender in 219 2016 using 4T1 cells, suggested that these Ru(IV) complexes are 220 readily washed out with phosphate buffered saline (PBS), and 221 raised doubts on whether the above deallylation reactions is intra222 cellular. In these experiments they describe the removal of an alloc 223 caging group in a luciferase substrate, which allowed for analyzing 224 the cellular reactivity by measuring fluorescent outputs [50]. 225 More recently, the group of Mascareñas et al., exploited the pres226 ence of 2-quinolinecarboxylate ligands in the ruthenium complexes 227 for the introduction of different cellular targeting appendages 228 (Fig. 2, complexes 6–8), such as phenyl-phosphonium groups that 229 may accumulate in mitochondria [51]. It is well known that triph230 enylphosphonium cations (TPP), because of exhibiting both a pos231 itive charge delocalized over three phenyl groups and a large 232 hydrophobic surface area, are able to accumulate in the mitochon233 drial inner membrane (IMM) driven by the membrane potential of 234 this organelle [52]. The authors demonstrated that despite the func235 tional complexity of mitochondria, it was possible to accumulate 236 active ruthenium complexes in this organelle by equipping the 237 ruthenium ligands with suitable arylphophosnium-type of delivery 238 vectors. For the visualization of the metal complexes, one of the 239 phenyl substituents of the TPP was replaced by a methylenepyrene 240 group resulting in a pyrene-phosphonium fluorescent directing 241 vector (complex 7,Fig. 2). Indeed, the presence of the phosphonium 242 group in the structure of the quinoline Ru ligand facilitates the 243 intracellular accumulation and the mitochondrial localization 244 (Fig. 4). 245 The presence of the pyrene group was observed to have a syn246 ergistic effect in the accumulation of the ruthenium complexes in 247 the mitochondria, promoting an increased concentration of 7in 248 this organelle up to 6-fold compared to the analog 6. Importantly, 249 the ruthenium complex presented a remarkable activity, which 250 was not only tested using standard fluorogenic probes, but also a 251 caged protonophore 1-(allyloxy)-2,4-dinitrobenzene (13) that 252 becomes an active mitochondrial uncoupler (14) only after in situ 253 Ru-promoted removal of the allyl protecting group (Fig. 5). Indeed, 254 using ruthenium complexes that do not accumulate in the mitoScheme 2. (Alloc)–amine protected uncaging mechanism proposed by Meggers and co-workers [49]. M. Martínez-Calvo, J.L. Mascareñas / Coordination Chemistry Reviews xxx (2018) xxx–xxx 5 CCR 112651 No. of Pages 23, Model 5G 8 January 2018 Please cite this article in press as: M. Martínez-Calvo, J.L. Mascareñas, Organometallic catalysis in biological media and living settings, Coord. Chem. Rev. (2018), https://doi.org/10.1016/j.ccr.2018.01.011 255 chondria, the uncaging reaction is much less efficient. This result 256 demonstrates for the first time that having an artificial catalyst 257 localized in a specific cell organelle can bring important biological 258 advantages, and sets the stage for future strategies for selective, 259 target associated drug activations. 260 In this article [51], the intracellular nature of the reaction was 261 further demonstrated by inductively coupled plasma mass spec262 trometry (ICP-MS) studies, which confirmed the presence of rea263 sonable amounts of ruthenium in the mitochondria, and by 264 incubating the ruthenium complexes prior to the substrates, to 265 ensure their accumulation inside the cells. 266 In early 2017, Volker and Meggers reported a new generation of 267 Ru complexes with improved stabilities and excellent catalytic 268 potential under bio-relevant conditions, even in blood serum 269 [53]. This new series of catalysts also possess a quinoline derived 270 ligand, 8-hydroxyquinolinate, in which the introduction of with271 drawing groups at the position 5 of the aromatic ring gave rise to 272 an increment in the catalytic activity (complex 9,Fig. 2). 273 All these data confirm that appropriately designed Ru com274 plexes are able to achieve highly interesting deallylation reactions 275 in biologically relevant complex aqueous mixtures and even in liv276 ing cells, in a bioorthogonal manner and without generating major Fig. 4. Imaging of the subcellular localization and catalytic activity of ruthenium complex 7(see Fig. 2). (a) Mitochondrial labeling with tetramethylrhodamine ethyl ester (TMRE) (red), (b) emission of cells incubated with the ruthenium complex (blue), (c) merging of (a) and (b), (d) fluorescence in cells pre-incubated with 7after addition of caged rhodamine 1, (e) merging of (a) and (d), (f) merging of (b) and (d) [51]. Fig. 5. In cellulo (HeLa) chemical rescue of the mitochondrial uncoupler 14 from the caged precursor 13 using ruthenium complex 7. (a) and (b) structures of the probes; (c) TMRE labeled cells after the incubation with 7(50 l M); (d) same experiment but with a previous incubation of the cells with ruthenium complex 7(50 l M) prior to the incubation of the allylprobe 13 (150 l M); the disappearance of the color indicates an efficient depolarization of the mitochondria; (e) TMRE labeled cells after incubation with 14 (500 l M) [51]. 6M. Martínez-Calvo, J.L. Mascareñas / Coordination Chemistry Reviews xxx (2018) xxx–xxx CCR 112651 No. of Pages 23, Model 5G 8 January 2018 Please cite this article in press as: M. Martínez-Calvo, J.L. Mascareñas, Organometallic catalysis in biological media and living settings, Coord. Chem. Rev. (2018), https://doi.org/10.1016/j.ccr.2018.01.011 277 toxicities. The application of this type of ruthenium complexes for 278 other type of reactions might open new, important opportunities in 279 research at the interface between organometallic catalysis and bio280 logical and cellular chemistry. In this context, a recent publication 281 by the group of Mascareñas et al., has demonstrated that complex 282 3can promote Ruthenium-Catalyzed Azide–Thioalkyne Cycloaddi283 tions (RuAtAC) in water, at room temperature and in the presence 284 of biomolecules (glutathione, aminoacids, peptides) [54]. The reac285 tion is also efficient in phosphate buffered saline (PBS) solution, 286 and in complex biological media such as cell lysates and fetal 287 bovine serum, and even in presence of living bacteria. Importantly, 288 the reaction is mutually compatible with the classical CuAAC, thus 289 providing for tandem bioorthogonal processes [54]. 290 2.2. Palladium 291 The use of palladium in biological environments is very appeal292 ing because of the well-demonstrated catalytic power of many pal293 ladium complexes [55], even in aqueous environments. 294 2.2.1. Protein modification 295 Pioneering work by the group of Davies et al. [56,57], demon296 strated that using Pd(OAc) 2 together with a 2-amino-4,6297 dihydroxypyrimidine ligands, commonly used in Sonogashira 298 Cross-Couplings in organic solvents [58,59], it is possible to 299 achieve Suzuki–Miyaura Cross-Couplings (Scheme 3)[60,61] on 300 appropriately functionalized proteins, and in biological buffers. In 301 vitro experiments showed that thiols like GSH might have a detri302 mental influence in the reactions. 303 They further explored the Suzuki–Miyaura Cross-Coupling reac304 tion for the 18 F labeling of bacterial protein SBL, reactions that 305 required the use of Pd(OAc) 2 as palladium source, and some of 306 the ligands L1–L4 indicated in Fig. 6 [62]. The same group was also 307 able to modify the cell-surface of Escherichia coli by using a Suzuki– 308 Miyaura Cross Coupling reaction on genetically ‘‘tagged” aryl 309 halide-containing porin channels, created through the incorpora310 tion of the unnatural amino acid pIPhe into OmpC protein mono311 mers [63]. Despite the undoubtable success of these pioneering 312 findings, these experiments were carried out in vitro or in the 313 cell-surface of E. coli, and avoiding the presence of free thiols which 314 seem to interfere with the Cross-Coupling reaction. 315 It is also remarkable the work of Lin and co-workers in the mod316 ification of an overexpressed modified ubiquitin (Ub) protein in 317 E. coli which incorporates an homopropargylglycine residue 318 (HPG), using a Sonogashira Cross Coupling reaction (Scheme 4) 319 [64]. They have expanded the application of the Cross-Coupling 320 reaction to the surface of mammalian cells, being able to modify 321 alkynyl-equipped proteins bound to the cellular membrane 322 [65,66]. These achievements meant a step forward on the develop323 ment of metal-promoted chemistry in living cells, however, it was 324 still limited to modify cell-surface-tethered biomolecules. 325 In 2014, the group of Chen et al., reported the use of discrete Pd 326 (II) catalysts to achieve chemical protein activations in living cells, 327 using a depropargylation or deallylation of caged lysines. In the 328 case of the propargylic (proc) systems, the reaction mechanism is 329 not completely clear, and could proceed via Pd II/IV or Pd 0/II cycles 330 (Scheme 5)[29]. 331 The effectivity of the Pd-promoted despropargylation and deal332 lylation reactions was monitored in vitro using the di333 propargylcarbamate–rhodamine probe 15 and bis-allylcarbamate 334 caged rhodamine (1), which become fluorescent (2) after removal 335 of the propargylcarbamate (proc) or allylcarbamate (alloc) pro336 tecting groups, respectively (Fig. 7). Among all the catalysts tested 337 in their report, Pd 2 (allyl) 2 Cl 2 and Pd(dba) 2 resulted the most effec338 tive for both types of uncaging in 5/95 DMSO/H 2 O using an Scheme 3. General mechanism for a Suzuki–Miyaura Cross Coupling [60,61]. Fig. 6. Representation of the Suzuki–Miyaura Cross-Coupling reaction for the 18 F labeling of SBL protein with Pd(OAc) 2 and the ligands L1–L4 [62]. M. Martínez-Calvo, J.L. Mascareñas / Coordination Chemistry Reviews xxx (2018) xxx–xxx 7 CCR 112651 No. of Pages 23, Model 5G 8 January 2018 Please cite this article in press as: M. Martínez-Calvo, J.L. Mascareñas, Organometallic catalysis in biological media and living settings, Coord. Chem. Rev. (2018), https://doi.org/10.1016/j.ccr.2018.01.011 339 equimolar ratio Pd/substrate (10 l M) (Fig. 7). Further experiments 340 in vitro using proc-lysine as substrate in PBS buffer, at 37 °C and 341 10% Pd loading, afforded reaction yields of 84% with Pd(dba) 2 342 and 82% with Pd 2 (allyl) 2 Cl 2 (Scheme 6). 343 Neither of these catalysts were able to efficiently uncage alloc344 lysine substrates under the same conditions, 26% and 22% yields 345 with Pd(dba) 2 and Pd 2 (allyl) 2 Cl 2 respectively. The proc-lysine 346 uncaging strategy was used for the chemical rescue of a nonScheme 4. Sonogashira Cross-Coupling labeling of a HPG-encoded Ub protein in E. coli [64]. Scheme 5. Mechanisms proposed for the depropargylation reactions promoted by either (a) Pd(II) or (b) Pd 0 complexes [29]. Fig. 7. (a) Uncaging reaction of probe 15 promoted by Pd catalysts and (b) Scheme representing a Pd-mediated activation of OspF bacterial enzyme by proc-lysine uncaging [29]. 8M. Martínez-Calvo, J.L. Mascareñas / Coordination Chemistry Reviews xxx (2018) xxx–xxx CCR 112651 No. of Pages 23, Model 5G 8 January 2018 Please cite this article in press as: M. Martínez-Calvo, J.L. Mascareñas, Organometallic catalysis in biological media and living settings, Coord. Chem. Rev. (2018), https://doi.org/10.1016/j.ccr.2018.01.011 612 turnover of the catalytic process. Thus far, the best results for cou613 pling a small azide with an alkyne in intracellular settings are 614 those reported byZimmerman et al. using Cu–MONPs. Considering 615 the enormous effect of the coordinating ligands in the stability, 616 reactivity, and toxicity of the copper complexes, one can foresee 617 that novel ligands might provide redox stable Cu(I) complexes that 618 could cross cell membranes efficiently, and therefore promote 619 intracellular CuAAC reactions in an effective way. 620 2.4. Gold 621 Gold complexes have been broadly studied in bioinorganic 622 chemistry and chemical biology for their bioactivity, mainly as 623 anticancer agents. Specially, cyclometallated gold(III) complexes 624 have been shown to elicit interesting pharmacological responses. 625 Complexes featuring CN, CNN and CNC-type of ligands have been 626 studied as protein inhibitors, DNA binders as well as promotors 627 of intracellular redox damage [98,99]. Albeit less studied, gold(I) 628 complexes with thiol, phosphine or NHC ligands have been also 629 identified as anticancer agents [98]. 630 However, the development of bioorthogonal transformations 631 promoted by gold complexes is still in a very early stage. Indeed, 632 the whole field of gold organometallic catalysis is quite young, 633 since it was not until recently that the reactivity of gold ions was 634 considered relevant [100–107]. The reactivity of Au(I) and Au(III) 635 complexes is associated to their carbophilicity, in particular to 636 the ability of these metals to coordinate and activate unsaturated 637 bonds. 638 Of course, most reactions catalyzed by gold complexes have 639 been reported in organic solvents, albeit some isolated transforma640 tions in aqueous media have also been described [108]. The trans641 lation of gold catalysis to biological media and cellular settings 642 does not seem obvious, however there have been several reports 643 on the development of sensing probes for gold ions in cellular enviFig. 17. (a) FRET probe activated by an Au 3+ promoted oxacyclization, (b) imaging of N2A cells treated with probe 28 (50 l M) only (i–iv) and the probe 28 for 30 min followed by AuCl 3 (250 l M) for 1 h at 37 °C (v–viii): images observed through: green channel (i) and (v) (500–575 nm); red channel (576–700 nm) (ii) and (vi); bright field images (iii) and (vii) and merged images (iv) and (viii). Scale bar: 10 l m. Adapted with permission from [110]. Copyright 2014 American Chemical Society. M. Martínez-Calvo, J.L. Mascareñas / Coordination Chemistry Reviews xxx (2018) xxx–xxx 15 CCR 112651 No. of Pages 23, Model 5G 8 January 2018 Please cite this article in press as: M. Martínez-Calvo, J.L. Mascareñas, Organometallic catalysis in biological media and living settings, Coord. Chem. Rev. (2018), https://doi.org/10.1016/j.ccr.2018.01.011 644 ronments, mainly Au(III) salts, that rely on gold-promoted 645 reactions. 646 In 2010, Kim and co-workers present some data on the viability 647 of using a gold-promoted cyclization of designed alkynyl probes to 648 provide fluorescent coumarin products (Scheme 8), however the 649 cell biology part of manuscript is presented in a very preliminary 650 way [109]. 651 In 2014, Ahn and co-workers reported another strategy for sens652 inggold salts incellsbased on agold(III)-promotedoxa-cyclizations, 653 withconcomitantringopening of arhodamine-lactamring(28).The 654 sensing system included a donor dye derived from 1,8655 naphthalimide that generates a fluorescence resonance energy 656 transfer (FRET) process with the rhodamine acceptor (29) 657 (Fig. 17). The authors demonstrated that the probe can be used to 658 detect gold salts purposely added to cells previously incubated with 659 the alkyne precursor [110]. 660 Recently, Tanaka and co-workers reported the development of a 661 Glyco–Au(III) complex that appears to be able to promote a gold662 catalyzed reaction in live mice. This Au(III) complex possess a 7663 diethylaminocoumarinlinkedtoawatercompatiblecyclometalated Fig. 18. (a) Au(III) cyclometallated complex 30 used in the studies; and (b) amide bond formation by alkynyl-ester activation in the mice liver, and intestine promoted by the conjugates Glyco–Au (Sia) and Glyco–Au (Gal) respectively. (Glycans, Sia = a (2 ?6)Disialo and Gal = galactosyl.) Reproduced by permission [111]. Copyright 2017, WileyVCH. Fig. 19. Role of the GSH on the assistance/inhibition on the Au-mediated alkynyl-ester activation. Reproduced by permission [112] Copyright 2017, Wiley-VCH. 16 M. Martínez-Calvo, J.L. Mascareñas / Coordination Chemistry Reviews xxx (2018) xxx–xxx CCR 112651 No. of Pages 23, Model 5G 8 January 2018 Please cite this article in press as: M. Martínez-Calvo, J.L. Mascareñas, Organometallic catalysis in biological media and living settings, Coord. Chem. Rev. (2018), https://doi.org/10.1016/j.ccr.2018.01.011 664 Au(III) complex via a short PEG linker (30,Fig. 18a). This conjugate 665 undergo a rapid translocation to target organs (i.e., liver, Glyco–Au 666 (Sia); intestine, Glyco–Au (Gal)) thanks to an interaction with 667 asparagine-linked glycans (N-glycans), and the resulting structures 668 arecapableofpromotingan amide bondformationbetweenfluores669 cent propargyl ester probes and nearbysurface-protein amines (e.g., 670 lysine side chains) (Fig. 18b) [111]. Despite the manuscript does not 671 provide details on the catalytic part of the work, and lacks controls 672 on the in vitro activity of the complexes, the work represents one 673 of the first applications of an Au(III) catalytic complex in live mice, 674 and thus opens new avenues on the potential applications of metal 675 catalysis in biological research. 676 More recently, Unciti-Broceta and co-workers, described the 677 preparation of gold nanoparticles (AuNPs, 30 nm) supported 678 within a PEG-grafted low-cross-linked polystyrene matrix (75 679 l m), and their use for promoting the removal of propargyl protect680 ing groups of a fluorogenic rhodamine [112]. It was possible to 681 carry out the reaction in serum-free biological medium. This Au– 682 resin was also successfully tested in cell cultures media, albeit 683 the transformations take place in the extracellular matrix; and 684 even in vivo, using zebrafish as animal model. 685 The mechanistic studies carried out by the group suggest that, 686 under physiological conditions, the deprotection reaction involves 687 the activation of the alkyne by the gold surface, and the nucle688 ophilic cooperative action of gold-tethered GSH. At higher concen689 trations of GSH, the reaction does not work, because the gold 690 surface is fully packed with GSH molecules which inhibit the 691 approach of the alkynes to the gold surface (Fig. 19). 692 Despite the progress in gold-promoted bioorthogonal reactions 693 in biological media is yet weak, and the biological applications of 694 gold catalysis are in their infancy, the distinctive reactivity of gold 695 species with respect to other metals suggest that we will see soon 696 new and relevant advances in the field. 697 2.5. Iridium 698 Most studies on the biological uses of Iridium organometallic 699 complexes have been focused on the area of bioinorganic and 700 medicinal chemistry, as well as in the development of imaging 701 agents. The use of iridium complexes as catalysts in biological set702 tings has been much more limited. The group of Sadler et al. has 703 pioneered interesting studies on the viability of using organoirid704 ium complexes as catalytic drugs inside cells, mainly for the 705 controlled alteration of the NADH/NAD + equilibrium [42,43,113– 706 115]. They have designed iridium complexes which are capable 707 of generating H 2 O 2 by catalytic hydride transfer from the coen708 zyme NADH to oxygen. Some of these organoiridium complexes 709 have even shown interesting anticancer potential owing to this 710 ability to change the redox status of the cell. 711 Recently, the group of Do et al., developed iridium complexes 712 which are able to promote aldehyde reductions in cell culture, 713 through hydride transfer processes mediated by NADH [116]. The 714 best results were obtained using iridium chloride complexes 715 equipped with pentametylcyclopendienyl groups and chelating 716 ligands like N-phenyl-2-pyridinecarboxamidate (Fig. 20a, com717 plexes 31–33). These iridium catalysts are fairly stable under phys718 iological conditions, tolerating moderate concentrations of 719 biological nucleophiles such as GSH. The complexes are able to 720 promote transfer hydrogenation processes in a versatile bioorthog721 onal way, which can be useful for the catalytic detoxification of 722 disease-causing agents. The reduction processes can be monitored 723 by fluorescence microscopy by using substrates such as BODIPY– 724 CHO (34), which upon reduction to its alcoholic form (BODIPY– 725 OH, (35)) experiment a change in the intensity of the fluorescence 726 emission (Fig. 20b) [117]. 727 The chemistry of Iridium catalysts in living cells has been essen728 tially confined to hydride transfer modifications. Thus, the real 729 challenge now lies on the translation to the biological medium of 730 other type of iridium-mediated processes, including processes 731 involving CAH activations. 732 2.6. Iron 733 In contrast to Pd, Ru, Au or Ir, iron is a transition metal very 734 commonly used by nature, also for catalytic processes, but mainly 735 in redox like transformations. The use of iron complexes to pro736 mote non-natural intracellular transformations with exogenous 737 substrates has been rather limited. Indeed, to the best of our 738 knowledge, there is only one example, published by Meggers 739 et al., describing the use of an iron(III) 5,10,15,20-tetraphenyl-21 740 H,23H-porphine (TPP) complex [Fe(TPP)]Cl (36) to promote the 741 reduction of the Rhodamine–bisazide 37 to rhodamine 110 (2) 742 (Fig. 21) in HeLa cells [118]. The reaction can be monitored by flu743 orescence, owing to the increase in the emission of green light of 744 the reduced probe. This experiment was also performed in vivo, 745 using nematodes and zebrafish as animal models, and it was Fig. 20. (a) Representation of the Ir(III) catalysts 31–33, the caged fluorophore 34 and the fluorogenic reduced probe 35, and (b) imaging of NIH-3T3 cells treated with (i) 34 (30 l M), (ii) 35 (30 l M), (iii) 34 (30 l M)/31 (20 l M), (iv) 34 (30 l M)/33 (10 l M), (v) 34 (30 l M)/IrCl 3 (20 l M), and (vi) 34 (30 l M)/31 (20 l M)/sodium pyruvate (10 mM). Sodium pyruvate was used to slow down the production of NADH which inhibited the hydride transfer from NADH to 53. Adapted by permission [117]. Copyright 2017, Wiley-VCH. M. Martínez-Calvo, J.L. Mascareñas / Coordination Chemistry Reviews xxx (2018) xxx–xxx 17 CCR 112651 No. of Pages 23, Model 5G 8 January 2018 Please cite this article in press as: M. Martínez-Calvo, J.L. Mascareñas, Organometallic catalysis in biological media and living settings, Coord. Chem. Rev. (2018), https://doi.org/10.1016/j.ccr.2018.01.011 746 possible to observe how the green emission was spread out 747 through the animal bodies. However, the authors later found out 748 that most probably the fluorescence is arising from in vivo meta749 bolic reduction of aromatic azides, not by the iron-mediated 750 reduction. 751 3. Artificial metalloenzymes 752 Not surprisingly, the more efficient metal-catalyzed transfor753 mations in aqueous and biological media are those promoted by 754 metalloenzymes, natural proteins equipped with metal cofactors 755 in their active site. However, in most of these reactions the metal 756 works either as a Lewis acid or as an electron transfer center, 757 and does not engage in organometallic mechanisms typically 758 found in other types of transition-metal catalysis (oxidative addi759 tions, reductive eliminations, migratory insertions...)[119]. 760 Therefore, along recent years there has been a great interest in 761 the development of metalloproteins that can achieve metal-based 762 transformations which are not present in nature. Most of the work 763 has been carried out in the context of asymmetric synthesis, albeit 764 some preliminary examples on the development of mimetic of nat765 ural enzymes capable of working in living environments have been 766 also developed. A number of detailed reviews in the area have been 767 recently published [35–40], and therefore we will not provide a 768 comprehensive review in this topic. Roelfes and co-workers pub769 lished several reports on the engineering of non-natural protein 770 catalysts by grafting non-proteinogenic amino acids capable of 771 binding a transition-metals, and therefore provide an active site 772 for different reactions. Thus, they built Bovine pancreatic 773 polypeptide–Cu complexes (bPP–Cu(II)) which are able to pro774 mote Diels–Alder and Michael addition reactions in water, with 775 enantioselectivities up to 86% [120]. Later, they grafted a new 776 active site onto the dimer interface of the protein LmrR by intro777 ducing bidentate phenanthroline and bipyridine ligands capable 778 of binding Cu(II) ions. The resulting metalloproteins allowed to 779 improve the enantioselectivity of the Diels–Alder reaction up to 780 97% enantiomeric excess (ee)[121]. In 2015 they also achieved 781 Friedel–Crafts reactions of indoles, in water, with good enantiose782 lectivity [122]. One year later, they were able to assemble a metal783 loenzyme in the context of the LmrR protein, incorporating a 784 metal-binding non-proteinogenic amino acid (2,20-bipyridin-5yl) 785 alanine (38), using gene expansion techniques. This represented 786 the first example of an artificial metalloenzyme with an in vivo 787 incorporated unnatural amino acid capable of binding a transition 788 metal ion and catalyzing an enantioselective reaction (Scheme 9) 789 [123]. 790 Additional pioneering work on the development of non-natural 791 metalloenzymes has been achieved by the group of Arnold, which 792 among other advances, has been able to build modified cyto793 chrome P450 proteins capable of promoting enantioselective 794 cyclopropanations [124], and aziridinations of olefins [125]. Fasan 795 and co-workers have also nicely contributed in this topic by the 796 use of cytochrome P450 variants for the oxidation of sp 3 CAH 797 bonds [126], and for the amination of sp 3 CAH bonds [127]. 798 The group of Hartwig and co-workers, presented a nice strategy 799 for the development of artificial metalloenzymes based on the 800 replacement of the native metals of the protein cofactors by noble 801 metals [128]. The designed systems elicited a totally different cat802 alytic activity, and the selectivity of the reaction could be modu803 lated by using directed evolution approaches. Thus, using heme 804 proteins as scaffolds and Fe–porphyrin IX [Fe–PIX] as reference 805 for the metal cofactor, they demonstrated that it is possible to Scheme 9. Representation of the modified LmrR metalloenzyme with an in vivo incorporated ligand 38, and reaction scheme of the benchmark catalytic Friedel–Crafts reaction between 39 and 40 to obtain 41. Reaction conditions: 9 mol% Cu(H 2 O) 6 (NO 3 ) 2 (90 l M) loading with 1.25 eq LmrR_LM_X (in monomer) in 20 mM MOPS buffer (pH 7.0), 150 mM NaCl, for 3 days at 4 °C[123]. Fig. 21. (a) Fe complex used for the reduction reaction; and (b) iron-catalyzed reduction of the azide 37 to generate fluorescent product 2[118]. 18 M. Martínez-Calvo, J.L. Mascareñas / Coordination Chemistry Reviews xxx (2018) xxx–xxx CCR 112651 No. of Pages 23, Model 5G 8 January 2018 Please cite this article in press as: M. Martínez-Calvo, J.L. Mascareñas, Organometallic catalysis in biological media and living settings, Coord. Chem. Rev. (2018), https://doi.org/10.1016/j.ccr.2018.01.011 806 substitute Fe by several noble metals (Fe(Cl)-, Co(Cl)-, Cu-, Mn(Cl)-, 807 Rh-, Ir(Cl)-, Ir(Me)-, Ru(CO)- and Ag-) to give novel type of metal808 loenzymes [128]. The methodology employed for the formation 809 of these artificial metalloenzymes involved express directly and 810 purify apo-PIX proteins lacking the entire heme unit, which is 811 reconstituted with the above derived metal cofactors containing 812 metals other than iron in a stoichiometric fashion. The strategy 813 involved minimal media lacking Fe to minimize the bio-synthesis 814 of hemin. This method indeed generated [M]–PIX–proteins with 815 the intact active site and with the cofactors bound at the native 816 PIX-binding site. These artificial metalloenzymes were evaluated 817 for asymmetric CAH insertion and cyclopropanation reactions 818 using carbenes, under biological relevant conditions (10 mM Tris, 819 pH 8.0 containing 8 vol.% MeCN) (Fig. 22). In the evaluation of 820 these artificial myoglobins ([M]–Myo) as catalysts, they found that 821 the [Ir(Me)–PIX] co-factor (42,Fig. 22) exhibited excellent perfor822 mances with eight myoglobin mutants for both reactions. In the 823 CAH insertion reactions were obtained selectivities up to an enan824 tiomeric ratio (e.r.) of 92:8 and with yields up to 97%. In the cyclo825 propanations they obtained e.r. up to 91:9 and a trans:cis ratio of 826 40:1. 827 Further research by the same group has led to improved ther828 mostable variant of cytochrome P450 from Sulfolobus solfatarius 829 (CYP119) containing the cofactor 42. The carbene insertion was 830 used again as model reaction. Several mutants from this artificial 831 metalloenzyme were studied in a variety of carbene insertion reac832 tions. In particular, mutants of the P450 enzyme Ir(Me)-CYP119 833 containing 42 as cofactor catalyzed insertions of carbenes into 834 CAH bonds with excellent yields and up to 98% ee. The quadruple 835 mutant of P450 Ir(Me)-CYP119-C317G-L69V-V254L (Ir(Me)-CYP836 Max) led to further improvements of both k cat and K M , creating 837 an enzyme with an efficiency that improved up to 4000-fold (k cat 838 = 45.8 min 1 ,K M = 0.17 mM, and k cat /K M = 269 min 1 mM 1 ) that 839 of the Ir(Me)-CYP119 system [129]. 840 While most of this work deals with the use of the designed 841 enzymes in asymmetric catalysis, preliminary attempts to build Fig. 22. (a) CAH insertion and, (b) cyclopropanation reactions promoted by [M]–Myo mutants (c) Structure of the co-factor 42 which provides better performance catalyzing CAH insertion and cyclopropanation reactions [128]. Scheme 10. Representation of the biot–Sav strategy for the formation of artificial metalloenzymes. Fig. 23. Biotinylated complexes derived from Ir (45 [130,131] and 46 [132]), Pd (47 [133]) and Ru (48 [134]), used for the assembly of metalloenzymes by Ward and coworkers. M. Martínez-Calvo, J.L. Mascareñas / Coordination Chemistry Reviews xxx (2018) xxx–xxx 19 CCR 112651 No. of Pages 23, Model 5G 8 January 2018 Please cite this article in press as: M. Martínez-Calvo, J.L. Mascareñas, Organometallic catalysis in biological media and living settings, Coord. Chem. Rev. (2018), https://doi.org/10.1016/j.ccr.2018.01.011 842 non-natural enzymes that can work in living environments are 843 starting to be published. Perhaps the more relevant study has been 844 recently reported by Ward and co-workers. This group has been 845 pioneered on the use of biotin–streptavidin (biot–Sav,Scheme 10) 846 interactions to build a variety of metalloenzymes equipped with 847 metal catalysts [40]. Many of these hybrids perform very well in 848 different type of transformations, including hydrogen transfer 849 reactions by the incorporation of a d 6 -piano chair complexes 850 within a host protein, [Cp ⁄ Ir(biot-p-L)Cl] (45)[130,131] and 851 [(Cp ⁄ -biot)Ir(L^L)Cl] (46)[132] giving rise to artificial transfer 852 hydrogenases (ATHase). The biot–Sav strategy has also been 853 applied to other bioorthogonal reactions such as the Suzuki– 854 Miyaura Cross Coupling (using biot–Pd 47 as cofactor), affording 855 an artificial Suzukiase for the synthesis of enantioenriched binaph856 thyls [133]. They have also built ruthenium-based metalloenzymes 857 using biot–Ru 48, which were able to promote a metathesis reac858 tion in the periplasm of E. coli (Fig. 23)[134]. 859 The group has demonstrated that embedding an organometallic 860 Iridium 45 complex within a host protein allows to overcome the 861 poisoning of transition metal in living cells by the presence of thi862 ols, mainly present in the form of GSH. The catalytic power of the 863 Sav-complex with iridium complex 45 was evaluated ex cellulo, 864 under different conditions, in the absence and in the presences of 865 the Sav mutant, cell lysates and E. coli culture and in the presence 866 of GSH neutralizing agents. While the free complex is capable to 867 promote the racemic conversion of 6,7-dimethoxy-1-methyl-3,4868 dihydroisoquinoline (49) to salsolidine (50) with excellent yields, 869 the presence of the mutant of the Sav S112A gave rise to excellent 870 yields and more than 80% ee (Scheme 11)[131]. 871 With regard to the metathesis reaction with organoruthenium 872 complex 48; the authors demonstrated that this species is nearly 873 inactive in cellulo, whereas the corresponding wild-type artificial 874 metalloenzyme biot–Ru–Sav peri endows the cell with metathesis 875 activity in the periplasm of E. coli (Fig. 24)[134]. 876 4. Conclusions and closing remarks 877 Achieving organometallic catalytic reactions of exogenous sub878 strates in the complex aqueous environment of living cells and tis879 sues is an enormous challenge. While the field is in its infancy, 880 there is a steady increase in publications reporting new type of 881 transformations in biological media and living cells. Therefore, 882 while up to 2010 the reports were mainly limited to copper883 catalyzed Click-type reactions, and only a few of them referring 884 to the inside living cells all, of them up to 20 publications; after 885 2010 we have counted around 100 articles dealing with other 886 metal-promoted transformations in biological media [30–33]. 887 Promoting intracellular reactions is particularly difficult owing 888 to the presence of a high concentration of components such as thi889 ols or amines, which can poison the metal and kill the catalytic 890 activity. Obtaining practical catalysts also require to deal with 891 other issues such as cellular transport and side toxicity. In the 892 future, it will be also needed to consider metal speciation, as well 893 as analyze turnover and reaction rates inside cells. 894 Furthermore, other questions such as catalyst confinement 895 within a specific organelle/environment, or the association of the 896 catalytic complex with specific targets, remain to be addressed. 897 Despite all these difficulties, the enormous possibilities offered 898 by organometallic chemistry, mainly because of the ligand vari899 ability that can be achieved, promises important future develop900 ments. Until know most of the advances have been essentially 901 limited to copper-promoted azide–alkyne annulations, and to 902 uncaging reactions triggered by ruthenium or palladium com903 plexes. There is therefore enormous room for developing other 904 type of transformations, such as cyclizations, coupling reactions, 905 annulations or CAH functionalizations, reactions that do not occur 906 in nature. For instance, in this context, recent work in the CAH 907 functionalization of nucleobases in aqueous media might lead to 908 future biological applications [135,136]. 909 Advances in ligand design might lead to good ratios between 910 reactivity and biological stability of the metal complexes, and even 911 avoid the need to use nanostructure formulations for their delivScheme 11. Asymmetric reduction promoted by embedded 45 employing the biot– Sav strategy [131]. Fig. 24. (a) Streptavidin is secreted to the periplasm by fusion to an N-terminal signal peptide from the outer membrane protein A (OmpA); (b) the biotinylated organoruthenium catalyst binds to the Sav forming the biot-Ru–Sav peri ; (c) catalytic bio-orthogonal metathesis reaction promoted by the biot-Ru–Sav peri metalloenzyme at the periplasm of E. coli. Adapted with permission from [134], copyright 2017 Nature Publishing Group. 20 M. Martínez-Calvo, J.L. Mascareñas / Coordination Chemistry Reviews xxx (2018) xxx–xxx CCR 112651 No. of Pages 23, Model 5G 8 January 2018 Please cite this article in press as: M. Martínez-Calvo, J.L. Mascareñas, Organometallic catalysis in biological media and living settings, Coord. Chem. Rev. (2018), https://doi.org/10.1016/j.ccr.2018.01.011 912 ery. Nanoparticles are not exempt of problems derived from the 913 protein corona effect and endosomal trapping; however, nanotech914 nology might also offer attractive opportunities in terms of trans915 port, toxicity control and spatio-temporal triggering of the 916 reactivity. 917 The expected progress in the field might therefore lead to 918 important future applications in biological and medicinal 919 chemistry. 920 Advances in this topic might allow for applications of the cat921 alytic power of the organometallic complexes in medicinal chem922 istry. Additionally, the development of artificial metalloenzymes 923 that can complement natural enzymes and therefore allow the 924 construction of an artificial metabolism is other of the future chal925 lenges in the area. 926 Competing financial interests 927 The authors declare no competing financial interests. 928 Acknowledgments 929 We are thankful for the financial support from the Xunta de 930 Galicia (Centro singular de investigación de Galicia accreditation 931 2016–2019) and the European Union (European Regional Develop932 ment Fund – ERDF). We also thank support given by the Spanish 933 grants sAF2013-41943-R and SAF2016-76689-R, the Xunta de Gali934 cia (GRC2013-041 and 2015-CP082), the ERDF, and the European 935 Research Council (Advanced Grant No. 340055). MMC thanks the 936 Ministerio de Economía y Competitividad for the Postdoctoral fel937 lowship (IJCI-2014-19326). 938 References 939 [1] R.H. 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