Organometallic catalysis in aqueous and biological environments: harnessing the power of metal carbenes

Rúa Jenaro de la Fuente, s/n – Campus Vida – Universidade de Santiago de Compostela – 15782 Santiago de Compostela – www.usc.es/ciqus Organometallic Catalysis in Aqueous and Biological Environments: Harnessing the Power of Metal Carbenes Sara Gutiérrez, María Tomás-Gamasa, José L. Mascareñas Peer reviewed version This is the peer reviewed version of the following article: Gutiérrez, S.; Tomás-Gamasa, M. and Mascareñas, J. L. (2022), Organometallic Catalysis in Aqueous and Biological Environments: Harnessing the Power of Metal Carbenes. Chem. Sci., 13: 6478-6495, which has been published in final form at https://doi.org/10.1039/D2SC00721E. This article may be used for non-commercial purposes in accordance with the Royal Society of Chemistry Terms and Conditions for Use of Self-Archived Versions. How to cite: Gutiérrez, S.; Tomás-Gamasa, M. and Mascareñas, J. L. (2022), Organometallic Catalysis in Aqueous and Biological Environments: Harnessing the Power of Metal Carbenes. Chem. Sci., 13: 6478-6495. doi: 10.1039/D2SC00721E Copyright information: © 2022 RSC. This article may be used for non-commercial purposes in accordance with the Royal Society of Chemistry Terms and Conditions for Use of Self-Archived Versions Organometallic catalysis in aqueous and biological environments: harnessing the power of metal carbenes Sara Guti´ errez, Mar´ ıa Tom´ as-Gamasa*and Jos´ e Luis Mascare˜ nas * Translating the power of transition metal catalysis to the native habitats of enzymes can significantly expand the possibilities of interrogating or manipulating natural biological systems, including living cells and organisms. This is especially relevant for organometallic reactions that have shown great potential in the field of organic synthesis, like the metal-catalyzed transfer of carbenes. While, at first sight, performing metal carbene chemistry in aqueous solvents, and especially in biologically relevant mixtures, does not seem obvious, in recent years there has been a growing number of reports demonstrating the feasibility of the task. Either using small molecule metal catalysts or artificial metalloenzymes, a number of carbene transfer reactions that tolerate aqueous and biorelevant media are being developed. This review intends to summarize the most relevant contributions, and establish the state of the art in this emerging research field. 1. Introduction Organometallic catalysis has revolutionized the way in which chemists conceive organic synthesis. The use of transition metals as catalysts transcended classical synthetic methodologies, and has allowed chemical transformations that otherwise would not be feasible. 1 Most of these organometallic reactions have been carried out in organic solvents, and usually under air and water-free conditions to avoid catalyst deactivation and the formation of side products. The fact that many organic substrates are not soluble in water has further averted the use of aqueous solvents in organic and organometallic reactions. However, there has been an increasing demonstration that many organometallic complexes and intermediates do tolerate the presence of water. Indeed, a huge number of watercompatible organometallic reactions have been recently developed. 2 Work in the eld has been further impelled by the growing interest in developing sustainable synthetic technologies, and by the observation that the presence of water can even accelerate some reactions. 3 These advances have led scientists to wonder if organometallic reactions could be carried out in biological and even living settings, as this could open new opportunities in cell biology and biomedicine. However, the translation of organometallic chemistry to biological habitats is not straightforward, and presents important challenges. First, a cellular milieu cannot be equated to a typical aqueous solvent, owing to its gel-like nature and intrinsic crowded environment. Furthermore, the presence of numerous biological components can promote the deactivation of the metal reagents or inhibit the catalytic cycles. Moving to living environments is further hampered by potential toxicity and transport issues, and by the low concentration of reagents and reactants. Nonetheless, with the advent of bioorthogonal chemistry, as coined by Bertozzi and coworkers, 4 an increasing number of organic reactions, including metal-catalyzed processes, have been demonstrated to work in biological and living environments. 5,6 A key discovery that sparked the eld is the well-known copper-catalyzed azide alkyne cycloaddition (CuAAC), paradigm of click chemistry, which is usually carried out with Cu(II) reagents and external reducing agents like ascorbate. Despite the intrinsic toxicity of these reagents, an appropriate tuning of the reaction conditions has allowed its use in cellular settings. 7–11 Its impressive chemoselectivity has also led to many applications for bioconjugation and post-translational modi- cation of proteins or nucleic acids. 12,13 The CuAAC entails a typical organometallic mechanism involving oxidative cyclometallation and reductive elimination steps, and it is a fundamental reference in the eld of biological organometallic catalysis. Another early (2006) report that has gained increasing recognition by the chemical biology community is the discovery by Meggers et al. that Ru(II) catalysts can promote the removal of alloc protecting groups in designed substrates, even in the presence of HeLa cells. 14 Although for several years there were not many new contributions in the area, in the last decade there has been an upsurge in reports dealing with transition metal-promoted reactions in Centro Singular de Investigaci´ on en Qu´ ımica Biol´ oxica e Materiais Moleculares (CiQUS), Departamento de Qu´ ımica Org´ anica, Universidade de Santiago de Compostela, 15705 Santiago de Compostela, Spain. E-mail: [email protected]; [email protected] Cite this: Chem. Sci., 2022, 13, 6478 Received 4th February 2022 Accepted 15th May 2022 DOI: 10.1039/d2sc00721e rsc.li/chemical-science 6478 |Chem. Sci., 2022, 13,6478–6495 © 2022 The Author(s). Published by the Royal Society of Chemistry Chemical Science REVIEW Open Access Article. Published on 16 May 2022. Downloaded on 6/9/2022 3:09:50 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online View Journal | View Issue biological media, and also in living organisms like bacteria. 15–20 Some of these discoveries have already found very relevant biological applications. 21–24 The reactions include uncaging processes, mainly those catalyzed by ruthenium or palladium complexes, such as the cleavage of N-allylcarbamates or O-allyl/ propargyl ethers, 14,25–33 ruthenium catalyzed bond-forming reactions (annulations between thioalkynes and azides (RuAtAC), 34,35 (2 + 2 + 2) cycloadditions 36 ) or isomerization of allylic alcohols, 37 gold-catalyzed hydroarylations, 38 and even palladium promoted Sonogashira 39 or Suzuki–Miyaura 40–43 cross couplings. Therefore, a wide range of organometallic transformations, from deprotection to cross-coupling, cyclization or cycloaddition reactions, have been demonstrated to be compatible with biological environments, provided appropriate metal complexes and probes are employed. What about metal carbenes? Can their reactivity be exported to biological environments? Metal carbene transfer reactions are among the most powerful and versatile transformations in catalysis and organometallic chemistry, and have been used for many synthetic applications. 44,45 Translating the rich reactivity typically exhibited by metal carbenes in organic solvents to aqueous and biological media, could open important new avenues in research at the interface between chemistry and biology. Indeed, in the last decade, there has been an increase in the number of reactions involving bioorthogonal metal-carbene transfer processes. In some cases, it has also been demonstrated that this chemistry can be performed in cells or in bacteria. In this review, we intend to cover the most signicant contributions in this topic, from bioconjugations and chemoselective modications of biopolymers to synthetic transformations of small molecules in biological settings, including bacteria or mammalian cells. 2. Catalysis involving metal carbene transfer reactions Carbenes are highly reactive chemical entities that possess a neutral divalent carbon atom with six electrons on its valence shell. According to the spin conguration of the two nonbonding electrons in the ground state, we can consider two types of carbenes: singlet carbenes, with both electrons occupying the sp 2 orbital with antiparallel spins, or triplet carbenes, with two electrons with parallel spins, occupying the sp 2 and the p z orbital (Fig. 1a). 46 A wide range of precursors have been employed for the generation of carbenes, such as tosylhydrazones, diaziridines, triazoles, cycloheptatrienes, alkynes or ynamides, among others. 47 However, the most widely used carbene precursors are diazo compounds, which are reasonably stable but can readily decompose to carbenes under mild reaction conditions. 44,48 The resulting free carbenes tend to be highly reactive, and therefore difficult to use in chemical transformations; however, upon appropriate coordination to transition metals they give metal carbenes, which present a more controllable and rich reactivity. The chemical properties of these metal complexes depend on the spin congurations of the carbene, and on the overlap with the metal orbitals. Different criteria have been used to classify metal carbenes. Historically, they have been categorized as Fischer and Schrock carbenes, depending on the metal complex and the type of substituents at the carbene; but in the context of this review, it is useful a classication based on the nature of the substituents adjacent to the reactive carbene centre. Acceptor/acceptor (A/A) and acceptor (A) carbenes are highly reactive due to the lack of stabilization of the electrophilic carbene centre. In contrast, the presence of donor groups stabilizes the metal carbene centre Fig. 1 (a) Electronic structure of carbenes. (b) Classification of metal carbenes based on the nature of the substituents adjacent to the carbene. (c) Some transformations of metal carbenes carried out in aqueous media. EDG ¼electron donating group; EWG ¼electron withdrawing group. © 2022 The Author(s). Published by the Royal Society of Chemistry Chem. Sci., 2022, 13,6478–6495 | 6479 Review Chemical Science Open Access Article. Published on 16 May 2022. Downloaded on 6/9/2022 3:09:50 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online and attenuates the reactivity. This is the case of donor/acceptor (D/A) carbenes, which have been widely used due to their good balance between reactivity and stability (Fig. 1b). 49,50 The rich reactivity of metal carbenes is mostly related to the charge distribution along the metal–carbon bond and the electrophilicity of the carbene carbon, allowing its reaction with a variety of nucleophiles. Within the broad spectrum of reactions enabled by metal carbenes, most common transformations include cyclopropanations of alkenes, 51–53 C–H functionalizations, 49,54–56 insertions into X–H bonds (X ¼O, N, S, Si), 57,58 the formation of ylides 59–61 (and subsequent rearrangements) or cycloaddition processes 44,61 (Fig. 1c). This reactivity is different from that exhibited by carbene precursors, usually diazocarbonyl compounds, which tend to decompose and/or engage in rearrangement or dimerization reactions. The reactions of metal carbenes with substrates exhibiting non-polar bonds (i.e. cyclopropanation of alkenes or insertions into C–HorSi–H bonds), tend to proceed through a concerted mechanism, 62 while in the case of insertions into polar X–H bonds a stepwise ylide mechanism might be operative. Beyond these general features, the characteristics of the transition metal complex play a key role in the mechanistic pathway. 58 Transition metal-catalyzed carbene transformations have been well studied in organic solvents, with rhodium, copper and iron being the most explored metals. It is important to note that besides carbene transfer reactions from diazo or related precursors, metal carbenes can also be used as catalysts, e.g. in metathesis processes. 3. Metal-promoted carbene transfer reactions in aqueous media The rich chemistry of metal carbenes has been explored essentially in organic solvents, and under water-free conditions, owing to the assumption that water might react with the carbene. However, as mentioned above, the reactivity of metal carbenes can be modulated by playing with the metal ligands and the substituents at the carbene atom, as well as with the type of substrates and reaction conditions; and thus, it can be made compatible with aqueous media. Indeed, in recent years there have been important contributions that demonstrate the viability of metal-mediated carbene transformations in aqueous media, and also in biologically relevant settings. Herein we present a summary of the most signicant developments. The reactions have been organized according to the type of substrate (small molecules or biopolymers) and, where relevant, to the type of transformation (cyclopropanation, C–H insertion, X–H insertion or ylide formation). Metal carbenes have also been used as catalysts for olen metathesis in aqueous and biorelevant environments. This topic has been covered in specic reviews, 63 and thus herein we will only mention some relevant examples performed in cellulo (Section 5.2). 3.1. Transformations of small molecules 3.1.1 Cyclopropanations. The cyclopropanation of styrene has been stablished as a benchmarking reaction in metal carbene transfer processes, with ethyl diazoacetate (EDA) being the most widely used carbene precursor. However, performing this type of reaction in aqueous media has only recently been addressed. The rst example of a metal-catalyzed cyclopropanation performed in the presence of large amounts of water was reported by the Nishiyama group in 2001. 64 The authors used EDA as carbene precursor, and a ruthenium complex exhibiting a water-soluble chiral ligand [hm-pybox: bis(hydroxymethyldihydrooxazolyl)pyridine] as catalyst, in a biphasic aqueous/organic milieu. The reaction yields the cyclopropane product with high levels of enantioselectivity (up to 94%, Fig. 2, trans/cis stereoselectivity up to 97 : 3). Curiously, the presence of water had a marked inuence in the enantioselectivity, which changed from 8% ee in pure THF to a 78% ee in THF/H 2 O mixtures. This result was attributed to a solvation effect of water around the hydroxy groups of the hm-pybox. In 2002 Charette and Wurz reported a different approach to perform similar cyclopropanations. They found that the combination of hydrophobic rhodium catalysts such as dirhodium(II) octanoate (Rh 2 (Oct) 4 ) with hydrophobic alkenes allows the formation of “small alkene/catalyst beads or micelles”in water. Then, a slow diffusion of EDA through the surface of the beads enabled a controlled formation of the desired cyclopropanated products in high yields (Fig. 2). Noticeably, watersoluble rhodium carboxylates (Rh 2 (OAc) 4 or Rh 2 (O 2 CCF 3 ) 4 ) were inefficient, leading to low yields. An asymmetric version of this reaction was also studied using ruthenium(II) and cobalt(II) chiral catalysts. 65 A family of catalysts that has been extensively employed for cyclopropanation reactions in organic solvents are metalloporphyrins, an interesting type of metal complexes that is also present in certain metalloenzymes with oxidative functions. In 2008, Simonneaux and coworkers prepared water-soluble ruthenium and iron porphyrins by the introduction of sulfonate groups, and used them as catalysts for the asymmetric Fig. 2 Metal catalyzed cyclopropanations. Top: General reaction scheme. Bottom-left: Asymmetric Ru-catalyzed cyclopropanation reported by Nishiyama. Bottom-right: Rh-catalyzed cyclopropanation reported by Charette and Wurz. 6480 |Chem. Sci., 2022, 13,6478–6495 © 2022 The Author(s). Published by the Royal Society of Chemistry Chemical Science Review Open Access Article. Published on 16 May 2022. Downloaded on 6/9/2022 3:09:50 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online cyclopropanation of styrene in water, with EDA as the carbene precursor (Fig. 3a). They obtained the desired products in yields up to 85%, with high diastereoselectivity (trans/cis up to 96/4) and good enantioselectivity for the trans isomer (83%). 66 In 2014, de Bruin and co-authors reported the rst example of size-selective cyclopropanation reactions using a soluble “molecular ship-in-a-bottle catalyst”. The bio-inspired supramolecular cage constituted by a cobalt-porphyrin catalyst encapsulated into a cubic M 8 L 6 cage (Fig. 3b) showed excellent activity in the cyclopropanation of styrene derivatives using water/acetone mixtures (5 : 1). 67 The reaction afforded the products in yields up to 88%, under mild reaction conditions (50 C) and low catalyst loadings (0.25 mol%, TONs up to 351). Remarkably, the porous cage-catalyst allowed for size selectivity; bulky substrates led to lower yields likely due to the slower migration through the cage pores. Another interesting approach based on the use of nanoreactors was reported in 2014 by van Hest et al., who immobilized chiral bis(oxazoline)–copper catalysts inside a polymersome membrane to perform asymmetric cyclopropanations of alkenes in water. Interestingly, only hydrophobic alkenes underwent the cyclopropanation, likely because of their ability to localize into the active site of the polymer. 68 All these results conrm the compatibility of specic metal carbenes with water, which even plays an important role in the reactivity and selectivity of the processes by eliciting hydrophobic effects. 3.1.2 Insertions into C–H bonds. Considering such compatibility, it is not surprising that other reactions mediated by metal carbenes have also been accomplished in water mixtures. In 2004, Afonso et al. observed that hydrophobic and sterically bulky a-diazoacetamides (such as N,N-diisopropylamide derivatives) can participate in intramolecular C–H insertion reactions in neat water, in presence of water soluble Rh 2 (OAc) 4 . The reaction was carried out at 80 C with turnover numbers (TON) of 883 for a total of 10 cycles. Interestingly, less hydrophobic substrates tend to give alcohols resulting from addition of water to the metal carbene (Fig. 4a). 69 These results suggest that hydrophobic metal carbenes somewhat avoid the reaction with water molecules, and prefer to undergo the C–H insertion. Further studies revealed that the use of more hydrophobic catalysts such as Rh 2 (Oct) 4 partially avoided or completely suppressed the hydroxylation reaction, even when less hydrophobic substrates were employed. 70 Another interesting metal-carbene mediated C–H insertion was proposed by the group of Ye, using ynamides as carbene precursors and gold catalysts. In the presence of an oxidant, the reaction generates a-oxo gold carbenes that can be trapped by indoles or anilines (Fig. 4b). The intermolecular reactions were carried out at 80 C using water as reaction media, providing the Fig. 3 (a) Water-soluble Fe and Ru porphyrins developed by Simonneaux and coworkers for asymmetric cyclopropanations in water. (b) Cobalt porphyrin “ship in a bottle”developed by de Bruin and coworkers, reproduced from ref. 67 with permission from Wiley-VCH GmbH, copyright 2014. Fig. 4 C–H insertion reactions in water. (a) Intramolecular insertion of rhodium carbenoids into aliphatic C–H bonds reported by Afonso and coworkers. (b) Functionalization of indoles and anilines by in situ generated a-oxo gold carbenes. © 2022 The Author(s). Published by the Royal Society of Chemistry Chem. Sci., 2022, 13,6478–6495 | 6481 Review Chemical Science Open Access Article. Published on 16 May 2022. Downloaded on 6/9/2022 3:09:50 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online C-alkylation products in excellent yields, aer 1–2 hours. 71 Remarkably, the presence of water suppressed undesired overoxidations of the gold carbene. 3.1.3 Insertions into X–H bonds. Insertion into X–H bonds (X ¼heteroatom) is a particularly interesting reactivity of metal carbenes to generate carbon–heteroatom bonds, and can also be achieved in the presence of water. In 2015, Simonneaux and coworkers reported the insertion of carbenes into N–H bonds of amino acid derivatives in aqueous media (Fig. 5a). The process is catalyzed by a water soluble iron porphyrin, FeTSPPCl ((5,10,15,20-tetrakis)-(4-sulfonato-phenyl)-porphyrin-iron(III) chloride), and was performed in citrate buffer at pH 10. In addition to the expected product, the reaction also produces small amounts of bis-inserted compounds. FeTSPPCl also proved to be an effective catalyst for site-selective modication of the terminal NH 2 group of insulin using EDA, and a PBS/ acetonitrile (7 : 4) mixture as reaction media, with 90% conversion aer 1 h. In this case, 10% of the double insertion product was detected. 72 In 2016, Sivasankar and coworkers reported an efficient synthesis of a-amino phosphonates in water via insertion of copper(I) carbenoids into the N–H bond of anilines under mild reaction conditions (Fig. 5b). The best results were obtained with (CH 3 CN) 4 CuClO 4 as catalyst, yielding the desired N–H insertion products in short reaction times (from 15 min to 2 h in most cases). 73 Later on, in 2017, they found conditions for the N–H insertion of carbenes exhibiting different electronic properties. AgOTf was the best choice for donor/donor carbene precursors, providing moderate yields aer 5 minutes of reaction, while for donor/acceptor carbenes containing aryl and ester groups, Pd 2 (dba) 3 led to the product in 90% yield aer 30 minutes. In the case of acceptor/acceptor carbenes, the most efficient catalyst was found to be [Ir(COD)Cl] 2 ,affording the N–H insertion product in 81% yield aer 12 h. The authors studied in more detail the reaction for acceptor/acceptor carbenes, and used their method to synthesize a series of anilinederivatives in good yields. However, in the case of aliphatic amines, such as benzylamine, and heteroaromatic amines like 2-aminopyridine, only traces amounts of the N–H insertion products were detected. 74 Fig. 5 N–H insertion reactions in water. (a) N–H insertion of iron carbenoids into amino acid derivatives catalyzed by a water soluble iron porphyrin in citrate buffer (CBS) reported by Simonneaux. (b) Carbene insertions into N–H bonds of anilines catalyzed by copper or iridium complexes reported by Sivasankar. (c) Iron catalyzed annulation of 1,2-diamines and diazodicarbonyl compounds reported by Lee. Fig. 6 (a) Gold-catalyzed tandem O–H insertion/cyclization in DMF/ H 2 O. (b) Doyle–Kirmse reaction of in situ generated sulfonium ylides. 6482 |Chem. Sci., 2022, 13,6478–6495 © 2022 The Author(s). Published by the Royal Society of Chemistry Chemical Science Review Open Access Article. Published on 16 May 2022. Downloaded on 6/9/2022 3:09:50 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online In 2016, Lee and coworkers reported an elegant strategy for the synthesis of quinoxalines, pyrazines and benzoquinoxalines through the annulation of 1,2-diamines and diazodicarbonyls in water at 70 C, using Fe(OTf) 3 as catalyst (Fig. 5c). The process is initiated by the insertion of an iron carbenoid into the N–H bond of the diamine, followed by cyclization and oxidative aromatization, to afford the desired heterocycles in excellent yields. 75 A relevant report by the group of Kwak in 2016 described the use of a Cu(I)-zeolite as a heterogeneous catalyst for the N–H insertion of a-diazoesters into substituted anilines. The reaction proceeds under mild conditions in a mixture of H 2 O/tBuOH (1 : 1). The Cu(I)-zeolite catalysts were stable and could be recycled, maintaining their activity over four reaction runs. 76 Gross and coworkers reported N–H insertion reactions of EDA and ethyl diazopropionate (EDP) on anilines in aqueous solutions, catalyzed by iron corrole conjugated albumins. The authors did not observe enantiomerically enriched products in any of the cases, and did not comment on the potential accelerating effect of the protein. 77 In some transformations, the insertion of water into the metal carbenes is the desired process. This is the case of the work of Wang et al., who developed a water insertion/ oxacyclization cascade of o-acetylenyl-substituted phenyldiazoacetates catalyzed by the gold(I) complex IPrAuCl (Fig. 6a). The process takes place through a sequence of gold carbene formation/water trapping and alcohol-alkyne 6-endo-dig cyclization. The transformation can take place in neat water providing mixtures (2 : 1) of 5-endo-dig and 6-endo-dig cyclization products; however, the best conditions consisted of using a mixture DMF/H 2 O (1 : 1) at 80 C for 24 h, providing 1H-isochromenes in moderate to good yields (64–82%). 78 3.1.4 Ylide formations. A powerful method for C–C bond formation is the [2,3]-sigmatropic rearrangement of sulfonium ylides generated in situ by reaction between a metal carbene and a sulde, also known as the Doyle–Kirmse reaction. This transformation has also been described in water, proceeding efficiently with Rh 2 (OAc) 4 (0.5 mol%) as catalyst, at room temperature, with different aryldiazoacetates and phenyl allyl sulde, affording the products in excellent yields (82–97%, Fig. 6b). 79 When phenyl propargyl sulde was used as reaction partner, the reaction was very slow, and a more hydrophobic catalyst such as Rh 2 (Oct) 4 was needed to obtain the products in high yields (87–95%). 3.2. Chemoselective reactions of peptides and proteins The above examples demonstrate that, in contrast to preestablished assumptions, the reactivity of metal carbenes can be harnessed in the presence of aqueous mixtures. This is especially relevant for their potential use to modify biological polymers like peptides or proteins, as most of them require aqueous solvents for an appropriate handling and solubility. As early as in 1966 it was shown that diazoketones, in the presence of copper salts, were able to inactivate pepsin, apparently due to the selective modication of a carboxylate residue in the active site of the protein. 80,81 However, truly designed protein modication reactions using metal carbenes were not reported until almost 40 years later. In 2004, Antos and Francis demonstrated the viability of using rhodium carbenoids for the selective modication of tryptophan side chains in myoglobin and subtilisin (Fig. 7a). The reaction was performed in water/ethyleneglycol (8 : 2, in the millimolar concentration range), using a-diazo esters and Rh 2 (OAc) 4 , and in the presence of hydroxylamine, providing a mixture of indole N–H and C–H insertion products. The addition of hydroxylamine dramatically enhanced the reactivity of the catalyst, presumably by binding to the distal rhodium of the bimetallic catalyst and stabilizing the reactive intermediates. 82 Subsequent studies using N-(tert-butyl)hydroxylamine as additive allowed modication of peptides and proteins (lysozyme and FKBP mutants) selectively on tryptophan residues exhibiting solvent-accessible indole side chains, at mild pH. 83 In 2013 Ball demonstrated the feasibility of performing selective cysteine modication under mild reaction conditions, using a N-(tert-butyl)hydroxylamine-containing buffer employed by Antos and Francis, Rh 2 (OAc) 4 and a biotin-tethered diazo substrate. The authors applied these reaction conditions to a mixture of proteins, and observed the modication of proteins with accessible cysteine thiols, while those with buried or oxidized cysteine residues remained unmodied. 84 Che et al. reported the rst example of a metalloporphyrincatalyzed carbene transfer for the selective modication of proteins. A designed water-soluble ruthenium glycosylated porphyrin catalyst [Ru II (4-Glc-TPP)(CO)] (1,4-Glc-TPP ¼mesotetrakis(4-(b-D-glucosyl)phenyl)porphyrinato dianion) was employed for different carbene transfer reactions in aqueous media (interand intramolecular cyclopropanation, Doyle– Kirmse reaction, N–H insertion). Interestingly, the catalyst proved to be effective for the selective alkylation of the Nterminus of peptides and proteins using a uorescenttethered diazo compound (Fig. 7b). However, when the protein presented free thiols (cysteines) in its structure, the insertion of the metal carbene took place into the S–H bond rather than in the terminal N–H bond. 85 Interestingly, Fischer carbenes have also been employed for bioconjugation reactions. Jaouen and coworkers performed a organotungsten labelling of bovine serum albumin using a Fischer carbene with the general formula L(CO) 4 W]C(OR 1 ) R 2 . 86 The authors observed a side-chain specic labelling in the amino group borne of some lysine residues, leading to stable aminocarbene adducts. On the other hand, Sarkar et al. modi- ed self-assembled monolayers (SAMs) on gold or glass with Fischer carbenes to immobilize protein A on surface, through reaction of pendant lysine residues with the electrophilic Fischer carbene (Fig. 7c). 87 Despite these successes, the reactions tend to be low yielding, and chemoselectivities are, in many cases, modest. As an alternative to these methods, Ball and coworkers developed an elegant strategy based on proximity-driven bioconjugations that combines molecular recognition with metallocarbene reactivity. This approach is based on the use of a dirhodium metallopeptide made of a rhodium(II) catalyst conjugated to © 2022 The Author(s). Published by the Royal Society of Chemistry Chem. Sci., 2022, 13,6478–6495 | 6483 Review Chemical Science Open Access Article. Published on 16 May 2022. Downloaded on 6/9/2022 3:09:50 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online a peptide that is able to recognize another specic protein (via a coiled-coil assembly, Fig. 7d). 88 Using this approach, the authors performed site-specic modication of peptides and proteins at physiological pH, and in biologically relevant buffers. 89–91 The use of biotin–diazo conjugates as reagents allowed for affinity tagging of the target proteins. They were even able to use designed metallopeptide catalysts to perform selective protein modications in E. coli lysate. Moreover, they developed a strategy for the site-specic functionalization of antibodies based on the use of a hexarhodium metallopeptide catalyst with affinity for the Fc fragment of the antibody. 92 Based on molecular recognition of the Fc region, this approach enabled the introduction of orthogonal alkyne handles into monoor polyclonal antibodies, opening the door to the quick production of antibody conjugates. 3.3. Chemoselective modication of nucleic acids Pioneering work by Gillingham et al. demonstrated the potential of metal carbenes to perform selective post-synthetic Fig. 7 (a) Bioconjugation of peptides and proteins based on the selective alkylation of tryptophan residues using rhodium carbenoids reported by Antos and Francis. (b) Bioconjugation of proteins based on the alkylation of the N-terminus. (c) Bioconjugation using Fischer carbenes on gold or glass surfaces. (d) Proximity-driven bioconjugation using rhodium metallopeptides developed by Ball for the site-specific modification of peptides, proteins and antibodies. The figure shows the structure of subtilisin Carlsberg ((a) PDB ID: 1SBC, tryptophans in blue) and RNase A ((b) PDB ID: 3A1R, N-terminus in blue). 6484 |Chem. Sci., 2022, 13,6478–6495 © 2022 The Author(s). Published by the Royal Society of Chemistry Chemical Science Review Open Access Article. Published on 16 May 2022. Downloaded on 6/9/2022 3:09:50 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online modications of oligonucleotides. In their rst report, in 2012, they used Rh 2 (OAc) 4 to catalyze the structure-selective N–H insertion of in situ generated rhodium(II) carbenoids into exocyclic amine groups of purine nucleobases under mild conditions. The reaction was carried out in aqueous buffers using donor/acceptor substituted carbenes at concentrations of 50 mM. This approach allows strategic targeting of solvent exposed nucleobases in double-stranded nucleic acids, as well as in single strands, bulge regions or overhangs (Fig. 8a, le). 93 Shortly thereaer, they demonstrated the effectiveness of Cu(I) salts, generated from copper sulfate and sodium ascorbate, to catalyze a similar insertion of donor/acceptor carbenes into the N–H bond of adenine in MES buffer. The diazo compound was further modied to contain an alkyne or azide handle, thus developing a tandem carbene insertion/CuAAC reaction (Fig. 8a, right). 94 Interestingly, the use of acceptor carbenes generated from adiazoesters or diazoacetamides and copper(I) led to chemoselective alkylation of the O 6 position in guanine (O 6 -G) in monoand oligonucleotides, attributed to the pre-coordination of the catalyst to the N 7 of guanine (Fig. 8b). 95 However, with complex oligonucleotides containing multiple chelation sites, reaction rates are low, likely due to unproductive catalyst sequestration. Very recently, Park and coworkers reported an elegant siteselective post-synthetic modication of oligonucleotides at unpaired guanosines. The use of a coordinatively saturated Rh(I) catalyst like [Rh(COD)Cl] 2 circumvented the chelation issues related to the deactivation of the catalyst observed by Gillingham, while maintaining the chemoselectivity towards base-unpaired guanosines in singleand double-stranded oligonucleotides. They exploited this feature and introduced Fig. 8 Nucleic acid alkylation using metal carbenes. (a) Structure-selective catalytic alkylation of DNA and RNA (left) and tandem carbene N–H insertion/CuAAC (right) reported by Gillingham. (b) Chemoselective alkylation at guanine O 6 -G using copper carbenes reported by Gillingham. (c) Site-selective functionalization of oligonucleotides using rhodium carbenes reported by Park, reproduced with permission from ref. 96, licensed under a Creative Commons Attribution (CC BY) license. It is attributed to Park, and the original version can be found here (https:// www.nature.com/articles/s41467-021-21839-4). © 2022 The Author(s). Published by the Royal Society of Chemistry Chem. Sci., 2022, 13,6478–6495 | 6485 Review Chemical Science Open Access Article. Published on 16 May 2022. Downloaded on 6/9/2022 3:09:50 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online heme, directly in bacteria. The strategy required the introduction of mutations within the heme binding pocket. The resulting “in-cell”assembled iridium complex promoted olen cyclopropanation reactions by EDA, and showed enhanced activity for aliphatic and electron-decient olens compared to the native heme enzyme. 150 Hartwig and coworkers recently demonstrated the viability of assembling an iridium containing heme metalloenzyme in bacteria, using the HUG system to transport the Ir(Me)MPIX cofactor into engineered cells expressing CYP119. They even demonstrated that it is possible to combine natural and arti- cial metalloenzymes in E. coli to afford cyclopropanated products from simple sugars (Fig. 19). 151 Very recently, they identied a second system for transporting iridium porphyrin into Nissle 1917 (a non-pathogenic E. coli strain), based on the presence of an outer-membrane receptor of these cells. 152 Metalloenzymes based on non-native protein scaffolds. As discussed above, it is possible to assemble articial metalloenzymes using a biotin–streptavidin recognition strategy. Ward demonstrated that a dirhodium complex tethered to a biotin moiety can interact with streptavidin proteins located in the periplasm of E. coli, and the resulting complex performed carbene transfer reactions in vivo. 137 The reactions are only effective in the periplasm, owing to the absence of thiols and other components that could deactivate the catalyst. In addition to carbene transfer reactions from diazo precursors, metal carbenes are also key reagents and intermediates in alkene metathesis processes, which can also be catalyzed by articial metaloenzymes. 101 Ward and coworkers designed a biotinylated Hoveyda–Grubbs second-generation catalyst (biot-Ru) that binds to streptavidin in the periplasm of E. coli, to give an effective articial metalloenzyme that promotes a ring-closing-metathesis (Fig. 20, le). A similar approach, based on the affinity of coumarin for albumin, was employed by the group of Tanaka to build ArMs that perform “in cell”metathesis reactions (Fig. 20, right). 153 6. Conclusions What seemed unfeasible at rst, achieving metal-catalyzed carbene transfer reactions in water mixtures, and even under the stringent conditions of a biological environment, has now become a reality. Exporting metal carbene chemistry to these reaction environments is possible, provided the substrates, catalysts and conditions are appropriately tuned. Considering the rich and versatile chemistry of metal carbenes, we foresee a bright future for further expanding the applications of these intermediates in chemical and cell biology, as well as biomedicine. Current challenges include, among others, the creation of versatile articial metalloenzymes that can perform chemoselective and bioorthogonal metal-carbene transfer reactions in mammalian cells, increasing rates and turnover with discrete metal catalysts, the development of metallocatalysts embedded in nanoscaffolds that facilitate the reactivity, or the development of biological applications. Author contributions JLM conceived the idea of the review and together with MTG supervised the project. SG prepared the dra, and MTG made the initial revision. JLM revised and edited the manuscript. All the authors participated in the discussion of the dras and gave approval to the nal version of the manuscript. Conflicts of interest There are no conicts to declare. Acknowledgements This work has received nancial support from Spanish grants (PID2019-108624RB-I00, RTI2018-093813-J-I00, R&C2020029150-I and ORFEO-CINQA network CTQ2016-81797-REDC), the Conseller´ ıa de Cultura, Educaci´ on e Ordenaci´ on Universitaria (ED431C-2021/25 and Centro Singular de Investigaci´ on de Galicia accreditation 2019–2022, ED431G 2019/03), the European Union (European Regional Development FundERDF corresponding to the multiannual nancial framework 2014–2020), and the European Research Council (Advanced Grant No. 340055). Figures have been created with https:// BioRender.com. Notes and references 1 J. F. Hartwig, Organotransition metal chemistry: from bonding to catalysis, University Science Books: Mill Valley, CA, 2010. 2 P. H. Dixneuf and V. 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