Intracellular Catalysis with Selected Metal Complexes and

Review Cite This: Chem. Rev. 2019, 119, 829−869

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Intracellular Catalysis with Selected Metal Complexes and Metallic Nanoparticles: Advances toward the Development of Catalytic Metallodrugs Joan Josep Soldevila-Barreda† and Nils Metzler-Nolte*,† †

Chem. Rev. 2019.119:829-869. Downloaded from pubs.acs.org by SWINBURNE UNIV OF TECHNOLOGY on 02/03/19. For personal use only.

Inorganic Chemistry IBioinorganic Chemistry, Ruhr University Bochum, Universitätsstrasse 150, 44780-D Bochum, Germany ABSTRACT: Platinum-containing drugs (e.g., cisplatin) are among the most frequently used chemotherapeutic agents. Their tremendous success has spurred research and development of other metal-based drugs, with notable achievements. Generally, the vast majority of metal-based drug candidates in clinical and developmental stages are stoichiometric agents, i.e., each metal complex reacts only once with their biological target. Additionally, many of these metal complexes are involved in side reactions, which not only reduce the effective amount of the drug but may also cause toxicity. On a separate note, transition metal complexes and nanoparticles have a well-established history of being potent catalysts for selective molecular transformations, with examples such as the Mo- and Ru-based catalysts for metathesis reactions (Nobel Prize in 2005) or palladium catalysts for C−C bond forming reactions such as Heck, Negishi, or Suzuki reactions (Nobel Prize in 2010). Also, notably, no direct biological equivalent of these transformations exists in a biological environment such as bacteria or mammalian cells. It is, therefore, only logical that recent interest has focused on developing transition-metal based catalytic systems that are capable of performing transformations inside cells, with the aim of inducing medicinally relevant cellular changes. Because unlike in stoichiometric reactions, a catalytically active compound may turn over many substrate molecules, only very small amounts of such a catalytic metallodrug are required to achieve a desired pharmacologic effect, and therefore, toxicity and side reactions are reduced. Furthermore, performing catalytic reactions in biological systems also opens the door for new methodologies to study the behavior of biomolecules in their natural state, e.g., via in situ labeling or by increasing/depleting their concentration at will. There is, of course, an art to the choice of catalysts and reactions which have to be compatible with biological conditions, namely an aqueous, oxygen-containing environment. In this review, we aim to describe new developments that bring together the far-distant worlds of transition-metal based catalysis and metal-based drugs, in what is termed “catalytic metallodrugs”. Here we will focus on transformations that have been performed on small biomolecules (such as shifting equilibria like in the NAD+/NADH or GSH/GSSG couples), on non-natural molecules such as dyes for imaging purposes, or on biomacromolecules such as proteins. Neither reactions involving release (e.g., CO) or transformation of small molecules (e.g., 1O2 production), degradation of biomolecules such as proteins, RNA or DNA nor lightinduced medicinal chemistry (e.g., photodynamic therapy) are covered, even if metal complexes are centrally involved in those. In each section, we describe the (inorganic) chemistry involved, as well as selected examples of biological applications in the hope that this snapshot of a new but quickly developing field will indeed inspire novel research and unprecedented interactions across disciplinary boundaries.

CONTENTS 1. Introduction 2. Biomolecule Transformations 2.1. Hydrogenation and Transfer Hydrogenation Reactions 2.1.1. Saturation of Lipids 2.1.2. Reduced Nicotinamide Adenine Dinuchleotide (NADH) Regeneration and Reduction of Pyruvate 2.1.3. Oxidation of NADH 2.1.4. Miscellaneous 2.2. Oxidation of Thiols 2.2.1. Glutathione (GSH) 3. Intracellular Transformation of Non-natural Molecules © 2019 American Chemical Society

3.1. Reduction of Carbonyl Groups 3.2. Reduction of Azide Groups 3.3. Reduction of Pt(IV) Complexes 3.4. Deallylation/Depropargylation Reactions 3.4.1. Molecular Transition Metal Complexes 3.4.2. Metal Nanoparticles 3.5. Miscellaneous 3.5.1. Rescue of Auxotroph Colonies 3.5.2. Deprotection of Amino Acids and Proteins

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Special Issue: Metals in Medicine

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Received: August 4, 2018 Published: January 8, 2019 829

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DOI: 10.1021/acs.chemrev.8b00493 Chem. Rev. 2019, 119, 829−869

Chemical Reviews 3.5.3. Deprotection of Carbohydrates 3.5.4. Deprotection of Genes 4. In Situ Labeling of Proteins and Synthesis of Molecules 4.1. Suzuki−Miyaura Reactions 4.2. Sonogashira Reactions 4.3. Metathesis Reactions 4.4. Azide−Alkyne Cycloaddition Reactions 4.5. Catalysis by Gold(III) Complexes 4.6. Miscellaneous Reactions 4.6.1. PEGylation of Proteins 5. Perspective Author Information Corresponding Author ORCID Notes Biographies Acknowledgments Summary of Complexes Discussed Cell Lines Mammalian Cells Bacteria Summary of Substrates Protecting Groups References

Review

also interfering with transcription.18,19 Ruthenium compounds such as RAPTA (Figure 1)20−22 or NAMI-A (Figure 1)23−28 were shown to have high affinity for proteins, although their exact mechanism of action is not known. Other compounds, such as the Au-based auranofin (Figure 1),1,29 can bind to cysteine residues in a variety of enzymes and inhibit their activity (most probably thioredoxin reductase, TrxR), or compounds such as the aspirin−hexacarbonyldicobalt conjugate (Figure 1)29,30 that inhibits cyclooxygenase activity due to the affinity of the aspirin ligand to the active center of the enzyme. While highly active, all the compounds mentioned to this point share a common particular characteristic; all react only one time with their targets. Whether this is in a defined 1:1 stoichiometry as in the case of enzyme inhibitors, or in a less well-defined stoichiometry as in the case of platin−DNA interaction is immaterial for our discussion here. Nevertheless, once the metal complex has interacted with a target molecule, it does not modify any other biomolecules any further. While undoubtedly successful, as proven by the clinical success of, e.g., cisplatin, this concept has inherent limitations. On one hand, high doses of the metallodrug might be necessary to produce the desired toxic effect on the cells or bacteria. On the other hand, and likely a consequence of the required high concentration, side reactions with other targets are common, inducing significant side effects and toxicity. Both situations, high doses and side reactions, have been a major concern over numerous years and traditional metallodrug design has concentrated on designing derivatives with higher specificity and affinity for the target.7,31−34 As an alternative strategy, there has recently been an interest in the development of metal complexes which are able to perform catalytic reactions inside biological systems (which are termed “catalytic metallodrugs”).7,35−41 Metal complexes in general have been known for a long time as potent catalysts, and this property is already used, inter alia, in the synthesis of fine chemicals and potent organic drugs. However, while the conditions for a metal-catalyzed transformation during the synthesis of a given chemical are typically highly optimized with respect to all parameters such as concentrations, solvent, or temperature, the situation is different for in vivo catalytic metallodrugs, which have to work in a given, complex environment. Ideally, the catalytic metallodrugs are target specific, stable under biological conditions, and display no or little toxicity while still maintaining significant catalytic activity. Such catalytic activity can broadly be defined as activation of a pro-drug or fluorophore or the capability of altering cellular components or processes. After completion of the catalytic transformation, the compound remains unchanged, allowing for a reaction with a new target molecule, thus having an amplification effect. Conceptually, this amplification leads to a reduction of the necessary dose of the metal compounds.7,35−41 In addition, there is potential for the reduction or elimination of side effects because the metallodrug by itself is ideally nontoxic and only affects the desired target molecule specifically. To date, there is already a variety of metal complexes which perform an activity as catalytic metallodrugs. For the purpose of this review, we wish to define four groups. First, metal compounds designed to perform reactions usually performed by natural enzymes (enzyme mimics).42−45 Of particular medicinal interest in this category are mimics of superoxide dismutase, but proteases and nucleases have also been studied

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1. INTRODUCTION Since the discovery of cisplatin and related platinum drugs, there has been a great interest in the development of metalbased drugs. In general, metal complexes offer a great diversity of geometries, containing up to six different ligands (in octahedral complexes), and the rational design of those ligands provides control over the reactivity and properties of the resulting complexes.1−11 For example, compounds such as carboplatin (Figure 1)12,13 or half-sandwich ruthenium ethylendiamine compounds (Figure 1)14−17 have a strong affinity for DNA and interfere with transcription. Similarly, IrIII or RhIII polypyridyl octahedral complexes can intercalate into DNA,

Figure 1. Chemical structures of metal-based drugs and drug candidates which react only one time with their targets 830


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for medicinal applications.35,46−50 Second, metal complexes capable of generating reactive oxygen species (ROS), sometimes with a known mechanism (e.g., ferrocene itself)51 and sometimes with unknown mechanisms.52 Also in this category are metal complexes that are capable of generating singlet oxygen inside cells upon irradiation with light (photodynamic therapy, PDT), which is a large field with metal compounds close to clinical application already.34,53−56 Third, metal complexes are capable of degrading proteins, peptides, DNA, or RNA. In this category are compounds such as NiII and CuII compounds containing the ATCUN moiety (an amino terminal peptide with His in position 3) which have been shown to cleave proteins57,58 and RNA.59−62 Also in this category are [CoII(cyclen)] derivatives, CuII-EDTA-biotin or Cu II -1,10-phenanthroline-arenesulphonamide derivatives, which can cleave peptide deformylase,63 amyloids,64−66 streptavidin,67 carbonic anhydrase,68 and other proteins depending on their ligand design. While tremendous success has been achieved in these first three groups with metal compounds through catalytic mechanisms mentioned, this review will not describe the advances in enzyme mimics,42,43,45,46,48−50 photodynamic therapy,34,54−56 ROS production,52,69−71 or biomolecules degradation35,38,72,73 because recent reviews already cover the topics very well. Instead, this review deals with a fourth group of catalytic metallodrug candidates, which has moved into the focus recently. These compounds are capable of performing reactions well-known in the organic synthesis repertoire, such as C−C cross-coupling reactions, cycloadditions, hydrogenation, and transfer hydrogenation reactions, thiol oxidation, or functional group deprotection reactions, inside living cells.36,38,39,41,74,75 It is also worth noticing that some of those catalytic reactions have been used for the in situ labeling of proteins, thus allowing the study of such proteins or their functionalization. We will cover metal-based catalysis that is performed by molecular transition metal complexes as well as by nanoparticles as appropriate. While we focus on the (inorganic) chemistry that is involved, giving conditions and commenting on limitations as far as they were described in the original literature, many selected examples of biological applications are included as well. We are aware of the fact that “catalytic metallodrugs” is a quickly developing and expanding field. Some aspects have been described in earlier reviews,7,35,37−41,43−46,50,55,71,76−84 but we hope that a comprehensive review at this moment (while always remaining a snapshot in a longer time frame) will inspire new researchers with new topics and visions to enrich this field.

also be obtained from other sources such as 2-propanol/KOH or formic acid/trimethylamine.85 Such transfer hydrogenation (TH) reactions are more broadly defined as reduction processes by which a catalyst can promote the transfer of a hydride ion from a donor to a molecule containing a multiple bond. In the early 1960s, initial reports of hydrogenation in aqueous media were presented.86 Currently, many common functional groups in synthetic chemistry are hydrogenated in water.86 TH reactions in aqueous media were not reported until the mid 1980s.87,88 Complexes such as [RuCl2(PPh3)3], [Cp*IrCl3], or [(η6-arene)Ru[TsDPEN)Cl] (4) were shown to reduce ketones, aldehydes, or imines in water or biphasic media (water/organic) using formate salt as a hydride donor.87,88 The use of aqueous media seemed to increase catalytic rates and allows for the use of more environmentally friendly solvents.87−89 Especially interesting is that the aqueous hydrogenation and TH reactions open the doors for the application of catalysis in biological systems, using either H2, different biomolecules as hydride sources, or formate as a nontoxic and biologically tolerated hydride source. 2.1.1. Saturation of Lipids. In 1985, Vigh and Joó studied a series of metal catalysts that can hydrogenate unsaturated double bonds in lipids.90−92 This method was intended to study the effect of lipid composition in the physical and biological behavior of cell membranes. Out of the numerous Ru and Rh phosphine and Pd complexes,90−92 catalyst [Pd(QS)2] (1) (QS = Sodium alizarinemonosulfonate) was established as the best catalyst and most of the subsequent work is performed using this one compound. In a typical experiment, cells were prepared in media and inserted in a hydrogenation vessel fitted with a septum. The vessel is evacuated and backfilled with H2. The catalyst (dissolved in water) is then added and the pressure of the reaction mixture increased. Saturation of lipids using catalyst 1 was shown to be very effective and selective for cis-double bonds in acyl chains, however, other chains are also saturated to a lower extent.47,91,93−97 Partial hydrogenation of cell membranes in different organisms and human cell types (Anacystis nidulans, Nicotina plumbaginifolia, Dunalille salina, human lymphocytes, or rat liver mitochondria) was achieved under controlled conditions.47,91,93−97 The degree of hydrogenation and the membrane affected (cytosolic or thylakoid) could be controlled by the reaction times.95 Interestingly, oversaturation of membrane lipids decreased cell integrity and viability. A number of papers have been published,47,91,93−97 however, the main focus is on the behavior of membranes and not the hydrogenation process. 2.1.2. Reduced Nicotinamide Adenine Dinuchleotide (NADH) Regeneration and Reduction of Pyruvate. In 1991, Steckhan et al. developed the first series of TH catalysts that were capable of regenerating NADH in aqueous media, pH 7 and 37 °C (i.e., under biologically relevant conditions), using high concentrations of formate as hydride source.98 Organometallic RhIII bipyridine (bpy) complexes were shown to reduce NAD+ efficiently with high turnover frequencies. The most active compound presented was [Cp*Rh(bpy)Cl]+ (2, Figure 2) with a TOF of 77.5 h−1 when performing the reaction using 0.5 M formate, 0.38 mM NAD+, and 25 μM complex 2 in biological conditions.98 NADH could then be used as a coenzyme for many enzymatic reactions such as the enzymatic reduction of ketones using LDH.99−101 In water, the complex undergoes hydrolysis to generate a reactive aqua

2. BIOMOLECULE TRANSFORMATIONS In this section, we will discuss reduction and oxidation reactions which have been performed inside cells. Such reactions offer a versatile tool for the study of cellular processes, regulation of redox environment, damaging the structural stability, disruption of metabolic processes, or induction of oxidative or reductive stress. 2.1. Hydrogenation and Transfer Hydrogenation Reactions

The use of transition metal complexes for the reduction of different molecules is one of the most studied catalytic reactions. Traditionally, molecular hydrogen would be used as a hydride source, activated by a metal catalyst and added to a double bond (hydrogenation). However, hydride anions can 831


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Figure 3. RuII catalysts for transfer hydrogenation reactions and regeneration of NADH. * Denotes a stereocenter.

formate at different concentrations. The eukaryotic cells were shown to tolerate high concentrations of formate, however, no significant effect was observed as a consequence of the coincubation with complex and formate.106 The work concluded that the catalytic reaction could probably be carried out in vitro, however, more active complexes would be necessary for a significant effect.106 At a later stage, a well-known TH catalyst was selected. Generally used in organic solvents, Noyori’s catalyst ([(pcym)Ru(TsDPEN)Cl], 4, Figure 3) is a RuII based complex that has shown very high TOF and enantioselectivity for the reduction of imines, carbonyls, or CC double bonds.111,112 Interestingly, complexes of the Noyori type were also active in aqueous media and even displayed higher TOF than in organic solvents.88 In addition, Hollman et al. reported in 2012 a modified heterogenic RhIII−Noyori type catalyst capable of reducing NAD+, proving that the complexes could potentially be used for regeneration of NADH.113 A series of neutral RuII half-sandwich Noyori type complexes [(η 6 -arene)Ru(sulfonamide)Cl] (η6-arene = bz, hmb, bip, or p-cym; sulfonamide = MsEn, TsEn or TfEn) were prepared (Figure 3).114 The complexes can regioselectively catalyze the TH of NAD+. Similarly to [(hmb)Ru(en)Cl]+ (3), the choice of arene had a major effect on the catalytic activity of the complexes, and turnover frequencies (TOF) decrease in the order bz > bip > p-cym > hmb, probably due to a combination of steric and electronic effects.114 As expected, the choice of the chelating ligand also had an important effect in that the more electron withdrawing substituents in the sulfonamide facilitated the hydride transfer.114 The TOF achieved where up to 7 times higher than those obtained previously for complex 3 under similar conditions (1.4 mM complex, 2.8 mM NAD+, and 35 mM formate at pD 7.2 and 37 °C). The most active compound was [(bz)Ru(TfEn)Cl] (5) with a TOF of 6.7 h−1 in D2O.114 In-depth study of the catalytic cycle was also performed using complex [(p-cym)Ru(TsEn)Cl] (6), including DFT modeling for the interactions of NAD+ with the metal complex.114

Figure 2. Catalytic cycle of complex [Cp*Rh(bpy)Cl]+ (2).

adduct. Formate can react with the aqua adduct to generate a formate−Rh intermediate that readily undergoes β-elimination generating a hydride adduct. Coordination of NAD+ via formation of an η3-Cp*Rh intermediate is followed by transfer of the hydride, giving rise to 1,4-NADH.102,103 On the basis of the work of Steckhan and Fish, different RuII, RhIII, and IrIII complexes have been developed for the chemical reduction of NAD+, however, [Cp*Rh(bpy)Cl]+ remains one of the most active compounds capable of reducing NAD+.104−107 In 2006, Sadler et al. proposed to exploit the TH properties to regenerate NADH inside cells.106 Using this approach, the concentration of NAD+ as well as the NAD+/NADH ratio could be altered, interfering with numerous processes such as energy regulation, DNA repair and transcription, or immunological functions.108,109 Alterations in the cellular redox status can play an important role in cell death, especially in diseases caused by an imbalance in oxidative stress such as cancer or Parkinson’s.108,109 Cancer cells are more dependent on redox regulatory systems and more sensitive to variations in the NAD+/NADH ratio (Figure 4).110 Initially, complexes [(η6arene)Ru(X,Y)Cl] PF6 (arene = indane (ind), hexamethylbenzene (hmb), p-cymene (p-cym); X,Y = ethylendiamine (en), bpy, or acetylacetone (acac)) were prepared and used to regenerate 1,4-NADH in a test tube (Figure 3).106 The complexes generated NADH under biological conditions in an NMR scale. A marked dependence on the arene ligand was observed, in which the stronger donors gave higher TOF. Unfortunately, the most active compound [(hmb)Ru(en)Cl]+ (3) gave a turnover frequency of only 0.82 h−1 when the reaction was performed using 1.7 mM complex, 3.3 mM NAD+, and 83 mM NaHCO2 at pD 7.2 and 37 °C, and therefore, the complexes can hardly be considered catalysts.106 The catalytic cycle was also studied, showing similar behavior to complex 2. A549 lung cancer cells were subsequently treated with complex 3, formate, and a combination of complexes and 832


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Figure 4. Proposed mechanism of action for catalytic NADH regeneration in cells (a).116 NAD+/NADH ratio in A2780 cells exposed to 1/3 × IC50 of complex 6 and sodium formate at different concentrations (0, 0.5, 1, and 2 mM), 24 h exposure (b) and at different time exposure (0, 2, 4, 12, 18, and 24 h) on the NAD+/NADH ratio, formate 2 mM (c).115 Reproduced with permission from ref 115. Copyright 2015 Nature https:// creativecommons.org/licenses/by/4.0/. Reproduced with permission from ref 116. Copyright 2015 Elsevier https://creativecommons.org/ licenses/by/4.0/.

Complexes of the type [(η6-arene)Ru(sulfonamide)Cl] (η6arene = p-cym or o-terpene (o-terp)) were then investigated for their anticancer activity in A2780 ovarian cancer cells.115 All the complexes displayed only moderate activity on their own, however, when the A2780 cells were coincubated with the complexes and formate, a significant decrease in cell survival was observed (Figure 4).115 This increase of antiproliferative activity was directly proportional to the formate concentration. The IC50 for complex [(p-cym)Ru(TsEn)Cl] (6, 13.6 μM) decreased to 1.0 μM when coincubated with 2 mM formate, a value comparable to that of cisplatin (1.2 μM) on the same cell line.115 When studying the cellular accumulation of the complexes in the presence and absence of formate, no differences could be observed. Thus, the increase of activity could uniquely be attributed to the combination of formate and complex.115 The NAD+/NADH ratio was also monitored when using different concentrations of formate, showing a direct correlation between the available hydride source (formate) and the decrease of NAD+. The NAD+/NADH ratio was also shown to decrease steadily over a period of 24 h when using 4.5 μM [(p-cym)Ru(TsEn)Cl] and 2 mM formate, allowing the calculation of an apparent TOF of 0.19 h−1 for the conversion of NAD+ to NADH in A2780 cells (Figure 4). The paper concludes that TH in cells is possible using Noyori type catalysts.115 Unfortunately, no correlation between activity in cells and in NMR could be observed, probably due to catalyst poisoning or side reactions in the cellular environment.

Tethered arene-N,N′ Noyori-type complexes (Wills catalyst, Figure 5) are well-known catalysts capable of reducing ketones

Figure 5. Wills catalyst for the efficient asymmetric transfer hydrogenation reaction of ketones. Anticancer activity was studied with enantiomerically pure compounds. * denotes chiral centers.

and imines with higher TOF and enantioselectivity than the original Noyori compounds.117,118 For this reason, the complexes’ anticancer activity toward various cell lines and the effect of formate was studied. The different enantiomerically pure tethered compounds displayed very low IC50 values which could be due to inhibition of microtubule polymerization. Interestingly, coincubation of A2780 cells with the compound and formate gave rise to a decrease on cell survival.119 However, the effect of formate was lower than in the case of nontethered compounds. In addition to the previously described complexes, a series of half-sandwich RhIII complexes of the type [(CpXRh(X,Y)Cl] 833


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(CpX = Cp*, 1,2,3,4-tetramethyl-5-phenyl-cyclopentadienyl (CpXPh) or 1,2,3,4-tetramethyl-5-byphenyl-cyclopentadienyl (CpXPhPh), X,Y = en, bpy, phenantroline (phen), and TfEn) were also prepared and their catalytic activity for transfer hydrogenation reactions under biologically relevant conditions was tested (Figure 6).116 Reduction of NAD+ to NADH was

enzyme peptide deformylase to formate. Co-administration of the compounds and N-formylmethionine to PC3 human prostate cancer cells (which overexpress peptide deformylase) gave rise to 20% reduction of cell survival compared to cells treated only with the complexes.121 In a last set of experiments, complexes R,R-[(p-cym)Os(TsDPEN)Cl] (R,R-9) or S,S-[(pcym)Os(TsDPEN)Cl] (S,S-9) (15 μM) were given to A2780 ovarian cancer cells in coadministration with sodium formate (2 mM), using an enantioselective assay kit for the detection of lactate. It was found that cells treated with R,R-9 show a significant increase of D-lactate, while the S,S-9 enantiomer showed an increase of L-lactate. This experiment proves that the reduction of pyruvate can be conducted inside cells, even with good enantioselectivity (Figure 7).121

Figure 6. Rhodium(III) complexes capable of reducing NAD+ under biologically relevant conditions.

achieved with higher TOF than those obtained for RuII complexes. Interestingly, the Noyori-type complex [(Cp*)Rh(TfEn)Cl] (7) showed higher catalytic activity than its RuII analogues but lower activity compared to the other RhIII complexes studied.116 The use of more sterically hindered, and electron withdrawing Cp rings (Cp* < CpXPh < CpXPhPh) increased the catalytic activity of the complexes, however, low solubility of CpXPhPh containing compounds renders the compounds nonuseful. The most active complex reported was [(CpXPhPh)Rh(en)Cl]+ (8), with a TOF of 24 h−1 in water.116 In addition to the NAD+ reduction, these complexes were also shown to be able to reduce pyruvate to lactate. However, this reduction showed lower TOF and occurred only after NAD+ was not available. The anticancer activity of the complexes in A2780 cancer cells was studied. Despite the fact that not all the complexes were active, cell survival decreased after addition of formate.116 In a recent report, Noyori-type OsII compounds were also studied for their transfer hydrogenation properties.120,121 The compounds were stable in water, even in their 16 electron state, and were capable of reducing acetophenone with >94% enantiomeric excess.120 The compounds were also tested for the reduction of pyruvate using formate as a hydride source and under biologically relevant conditions, giving rise to Llactate or D-lactate with a high enantiomeric excess.121 When studying the complexes’ effect on A2780 cells, the compounds were shown to accumulate in the cytosol and the membrane, with IC50 values of all compounds 100 μM) (Figure 10).136 However, NAD+/ NADH ratios are still affected. This is due to the extent of NADH oxidation and the molecules that are reduced with the formation of the Ir−H species. In recent work, Do et al. have studied the possibility of using this nontoxic Ir complex as a chemosensitizer which increases the levels of ROS in cells.136 The cells are cotreated with the iridium catalyst and with wellknown Pt-based chemotherapeutics like cisplatin or carboplatin, thus obtaining a decrease of cell survival of up to 50% in some cell lines. In the paper, levels of ROS and NAD+/NADH ratios were studied in the presence and absence of Ir catalyst or platinum drugs, demonstrating that the Ir compounds have a major effect on the redox homeostasis of the cell. Also interesting is that this approach provides a degree of selectivity toward cancer cell because healthy cells were proven to sense the effect of Ir complexes to a much lower extend.136 When looking at the work of Sadler and Do, it is evident that oxidation of NADH itself does not induce cell death but causes an imbalance on the redox state of the cell. However, when this imbalance occurs simultaneously to an increase of ROS, or in the presence of other anticancer drugs that interfere with 836


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Figure 11. Complex [Cp*Ir(biot-p-L)Cl] (20)

Figure 12. Scheme for the role of GSH and effect of catalytic oxidation of GSH in cells. Upon internalization of the catalyst and reaction with GSH, the concentration of GSH decreases dramatically. GSH performs numerous functions such as detoxification or maintaining redox homeostasis. Lower concentrations of GSH affects those functions. Reproduced with permission from ref 38. Copyright 2015 Elsevier https://creativecommons.org/licenses/by/4.0/.

cofactors. However, no in-cell reaction has been tested to date.141−144 Do et al. have recently also studied the effect of different biologically relevant components on selected known IrIII and RuII catalysts capable of oxidizing NADH (Figure 10).145 Complexes such as 14, 15, 16, 17, 18 [(Cp*)2Ir2(4,4′,6,6′tetrahydroxobipyrimidine)Cl2]2+, or [(hmb)Ru(phen)Cl]2+ (2 mol %) were dissolved in tBuOH/phosphate buffer (2:8) containing 1.2 mol equiv NADH and 1 equiv of benzaldehyde. After 24 h at 37 °C, only compound 17 and derivates gave high yields (81−91%) for the reduction of benzaldehyde. The reaction could also be performed using formate as a hydride donor instead of NADH. Despite the lack of a TH reaction, complexes 14 and 15 were also shown to form hydrides (with characteristic 1H NMR signals at −11.5 and −15.3 ppm, respectively) under the same conditions, thus maybe the hydride transfer could occur with different substrates.145 When performing the reaction (2 mol % catalyst 17, 1 equiv benzaldehyde, and 1.2 equiv NADH in tBuOH/phosphate at 37 °C) with added common biomolecules (1 mM) such as GSH, glucose, ascorbic acid, or nucleobases, good TH conversions were still achieved.145 In cell culture media, the reaction also occurs, however, the yield decreases to 40−50% after 15 h. The reaction was also studied for different aldehydes and ketones, reducing mainly the aldehydes (both aromatic and aliphatic) to their corresponding alcohols. In the paper, it is also proposed that 17 can act as an aldehyde dehydrogenase mimic and be used to reduce low-molecular-weight α,βunsaturated aldehydes formed in lipid peroxidation.145

drugs like cisplatin, CuII-triapine, or other CuII-thiosemicarbazones.149−151 Catalytic depletion of GSH using metal complexes has also been studied.36,78,82,152 These compounds were intended to generate an imbalance in redox homeostasis, driving cells into apoptosis by oxidative stress. However, little literature on the topic is available, and in many cases, GSH oxidation occurs unintentionally as a side reaction and not as the main mechanism. Initial reports on the topic can be traced to 2008, when Sadler et al. presented a series of RuII half sandwich complexes containing azopyridine chelating ligands (Figure 13).153 In this paper, the complex [(bip)Ru(azopy-NMe2)I]+ (21) was shown to hydrolyze and form a GS adduct in aqueous media. In addition, when 10 mol equiv GSH were reacted with 100 μM complex 21 in phosphate buffer (10 mM) at pH 7.2 and 37 °C under an inert atmosphere for 24 h, the formation of GSSG (oxidized GSH) was observed via 1H NMR, HPLC, and ESI-MS. The reaction was catalytic but had only a low TOF (0.37 h−1). In further experiments, no H2 or H 2O 2 were observed, however, formation of O 2 was detected.153 A catalytic cycle was also proposed involving a ligand-based redox reaction. To confirm the reaction occurred in cells, A549 human alveolar epithelial adenocarcinoma cancer cells were treated with the complex, and the formation of reactive oxygen species (ROS) was detected by fluorescence.153 Despite the observed increase of ROS, GSH, or GSSG were not quantified independently. In the following years, another series of Ru complexes containing redox active diamine ligands (o-phenylendiamine, o-pda /o-benzoquinonediimine, o-bqdi ) were studied (Figure 13).154 Complexes such as [(p-cym)Ru(o-bqdi)I]+ (22) were shown to hydrolyze in water at 37 °C. However, because of the presence of O2, the compounds also oxidize to [(p-cym)Ru(opda)I]+. In the presence of 15 mM GSH, the oxidation of the complex was followed by regeneration of 22 and GSSG formation.154 The reaction was followed by UV−vis and 1H NMR until depletion of GSH and regeneration of 22. Despite the fact that no claims for catalytic activity were made, the compounds were able to oxidize GSH to GSSG and be regenerated by O2. Cytotoxicity tests on A2780 and A549 cells were performed, showing that complexes containing the oxidized ligand were active but not those containing the

2.2. Oxidation of Thiols

Sulfur containing molecules play a very important role in biological systems, from maintaining the structure of proteins or enzymes to detoxification or antioxidant action. Of particular interest is cysteine, a natural amino acid capable of oxidation to form disulfide R−S−S−R bonds. Many metal complexes have been shown to interact with cysteine residues in proteins or peptides, either as their biological target or as a detoxification pathway. For example, cisplatin is known to bind irreversibly to glutathione (GSH) and is expelled out of the cells.146,147 2.2.1. Glutathione (GSH). GSH is one of the more abundant low molecular weight thiols in cells.148 It plays an important role in controlling and maintaining the redox homeostasis of cells by reducing elevated levels of reactive oxygen species (ROS) (Figure 12).148 GSH is also known to act as a detoxifying agent, as in the case of cisplatin (Figure 12).146−148 The use of drugs capable of depleting GSH has been studied in combination therapy to enhance the effect of 837


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Figure 13. Complexes capable of catalyzing oxidation of GSH.

obtained with no signs of decomposition. In later studies, it was also found that the compounds react mainly with cysteine and GSH even in the presence of other components. In addition, a correlation between lipophobicity of the ligands and toxicity could be established.160 Despite the fact that no direct correlation between GSH oxidation and cytotoxicity could be drawn, it was concluded that the toxicity of the complexes was probably, at least partially, due to their ability to catalytically oxidize GSH.160 Interestingly, when preparing RhIII and IrIII analogues of the thiolato-bridged dimers, high toxicity was still obtained, albeit somewhat lower than for the RuII analogues. However, these compounds exhibited poor catalytic activity (TOF 0.34 h−1 for complex [(Cp*)2Rh2(μ-SC6H4-p-CH3)3]+ (26)). Again, the cytotoxicity could not be correlated with catalytic activity.161 In the same year, Therrien, Furrer et al. published their study on two supramolecular Ru II compounds, [(pcym)6Ru6(R)3(tpt)2] where tpt = 2,4,6-tris(pyridine-4-yl)1,3,5-triazine and R = 2,5-dihydroxy-1,4-benzoquinonato (dhbq, complex 27) or 2,5-dihydroxy-1,4naphthoquinonato (dhnq, complex 28) (Figure 13).162,163 The compounds form a “cage” capable of encapsulating small molecules which can be later released. The compounds were stable in water, but in the presence of Cl− or CO3− slow decomposition was observed. Similarly, the compounds also decompose in the presence of Arg, Lys, and His to form the corresponding amino acid adducts. Interestingly, when complex 27 or 28 was reacted

reduced ligand. No further analysis on GSH/GSSG or ROS was performed.154 Recently, a series of iridium compounds of the type [(CpX)Ir(N,N′)SH]+ (CpX = Cp* or CpxPhPh, N,N′ = bipyridine or phenantroline) were also synthesized and studied for their anticancer activity (Figure 13).155 Compounds containing Cp* ligands displayed moderate activity toward A2780 human ovarian cancer cells, while [(CpxPhPh)Ir(bpy)SH]+ (23) and [(CpxPhPh)Ir(phen)SH]+ (24) showed IC50 values lower that cisplatin. Reactivity studies with GSH showed that none of the complexes is capable of forming GSH adducts, although both compounds 23 and 24 are capable of oxidizing GSH to GSSG.155 Despite the catalytic oxidation of GSH by 23 and 24, no proof of such reaction inside cells has yet been provided. Süss-Fink, Dyson, and Therrien published a series of RuII thiolato-bridged compounds with very high antiproliferative activity toward cancer cells and specifically cisplatin-resistant cell lines (Figure 13).156−160 When studying the stability of the complex [(p-cym)2Ru2(μ-SC6H4-p-CH3)3]+ (25) in the presence of different biologically relevant molecules like amino acids, glucose, or nucleosides, the compounds were shown to be very stable. Surprisingly, when using cysteine or glutathione, the compounds were capable to oxidize GSH to GSSG. The TOF of the reaction with GSH (1:100 complex:GSH) at pD 7, 37 °C, and 50 mM NaCl was 7.4 h−1.157 After the catalytic reaction, the compound could be 838


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Figure 14. Activation of Bodipy-CHO via reduction with 17 (a). Confocal microscope images of NIH-3T3 cells treated with Bodipy-CHO, Bodipy-OH, and Bodipy-CHO + 17 (b). Reproduced with permission from ref 164. Copyright 2017 American Chemical Society.

series of compounds containing aldehydes where synthesized which, upon reduction, become fluorogenic probes used for cellular imaging (Figure 14). The main example used was Bodipy-CHO. When Bodipy-CHO was treated with complex 17 (2 mol %) and NADH (2.1 equiv) at 37 °C, 20% of the substrate was reduced after 24 h.164 However, the fluorescence emission increased about 2-fold after only 4 h. Similarly, a fluorescence enhancement of up to 1.6 times after 2 h was observed when NIH-3T3 mouse embryo fibroblast cells (Figure 14) and A549 human alveolar epithelial adenocarcinoma cells were treated with Bodipy-CHO (30 μM) and then exposed to complex 17 (20 μM).164 No increase in fluorescence could be observed when using a less active catalysts (complex 15) or when depleting the intracellular NADH pool. Importantly, the combination of Bodipy-CHO and IrIII catalysts or either component on its own had no detrimental effect in the cells during the course of the experiments.

with 6 mol equiv of cysteine, formation of cystine (the oxidized dimer of cysteine) was observed. Complex 27 does not decompose in the presence of cysteine,162 however, complex 28 forms stable dimeric structures [(p-cym)2Ru2(cystine)]+. This behavior can also be observed with GSH.162 The compounds were capable of oxidizing Cys and GSH catalytically, which seems a reasonable suggestion for their mechanism of anticancer activity.

3. INTRACELLULAR TRANSFORMATION OF NON-NATURAL MOLECULES For many years, there has been an intense effort in developing methods for triggered activation of pro-drugs in cellular environment using either light, cellular components, or salt concentrations. However, recently, there has been an increasing interest in the development of catalytic reactions that can promote the activation of prodrugs in cells. Those reactions not only allow the site-specific activation of prodrugs in a particular area but also allow the use of minimal amounts of activator (catalyst). This research could lead to new mechanisms of action, the use of lower doses of toxic drugs, and an increased control over target selectivity. In the following section, we will discuss the advances achieved in selective transformation (usually described as an activation) of prodrugs and fluorophores inside cells. In fact, while the ultimate goal is catalytic prodrug activation, very often the decaging of fluorophores is chosen as the model reaction to prove intracellular activity. In addition, some applications of these methods for the rescue of auxotrophic cells, activation of proteins, and labeling of cellular components will also be discussed.

3.2. Reduction of Azide Groups

Because of their inertness in biological systems, azide functional groups offer an elegant scaffold for the development of nonactive prodrugs, which could be activated upon reduction to their corresponding amine. Following this idea, Meggers et al. published a series of metal−porphyrin compounds and studied their ability to reduce azide groups.165 Interestingly, only iron analogues were shown to catalytically reduce azides. To evaluate the azide reduction potential under biological conditions, an azide-containing rhodamine 110 derivative (N3RH) was designed. While N3-RH showed no fluorescence, highly fluorescent rhodamine 110 (fluorophore, RH) is formed after reduction. When N3-RH was treated with 5% [Fe(TPP)Cl] (29) in phosphate buffer (20 mM, pH 7.2) at 37 °C, no reduction occurred.165 However, upon addition of L-cysteine (5 mM), complex 29 was able to reduce the azide in 13% yield

3.1. Reduction of Carbonyl Groups

In a recent paper, Do et al. investigated the possibility of using Ir transfer hydrogenation catalysts for the bioorthogonal activation of target molecules in cells.164 In their work, a 839


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(Figure 15). Increasing concentrations of reducing agent (cysteine, GSH or ascorbate) resulted in increased fluorescence.

Liu et al. described the reduction of azide groups by photoactive [Ru(bpy)3]Cl2 (30) when irradiated with visible light.166 In the paper, different DNA-aryl azides are reduced to their corresponding amines using 30 and light in organic solvents and with tertiary amines as hydrogen donors.166 However, the addition of Hantzsch esters increases the reaction rate and conversion. When the reaction was performed in aqueous media, the Hantzsch esters can be replaced by NADH with only small effects in the catalytic reaction.166 The combination of tertiary amine/NADH can be replaced by ascorbate with no detrimental effect.166 More importantly, the reaction could be performed in the presence of many functional groups without any side reaction occurring.166 Different DNA oligonucleotides, oligosaccharides, or proteins with azide groups were used to test the activity of 30 and ascorbate with successful results.166 In a last experiment, a DNA oligonucleotide linked to a carboxylic acid was protected with an azidophenyloxycarbonyl group. Upon reduction of the azide with 30 and ascorbate, a 1,6-elimination occurs, releasing the free carboxylic-oligonucleotide.166 Following the same approach, Winssinger et al. synthesized a nonfluorescent quinazolinone precipitating dye protected with an azidophenyloxycarbonyl group (azoc-QPD). Upon irradiation (1 W LED light, λ = 455 nm) in the presence of 30 and

Figure 15. Reduction of N3-RH using 29 and GSH.

An impressive 28-fold increase in fluorescence was also achieved after only 10 min when HeLa cells (cervical cancer cells) were incubated with 100 μM N3-RH (25 min), washed, and subsequently treated with 10 μM complex 29.165 Cells treated only with N3-RH showed no fluorescence. Unfortunately, when N3-RH was used in nematodes or zebrafish, reduction of the azide occurred naturally, and therefore this approach was not appropriate for use in vivo.165

Figure 16. Azide reduction using 30 (a), Activation of azoc-QPD using 30 (b). Structures of complexes 30 and [Ru(bpy)2(EGFR inhibitorphen)]PF6 (c). Transfected and nontransfected HEK293T cells treated with 30 and azoc-QPD (d).167 Reproduced with permission from ref 167. Copyright 2015 Royal Society of Chemistry. 840


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ascorbate, the dye (fluorophore) is released (Figure 16).167 This catalytic reaction can also be conducted using variants of the complex containing bpy- or phen-type ligands.167 The ruthenium complex [Ru(bpy) 2 (EGFR-inhibitorphen)]PF6 was prepared in order to target specifically EGFR (a tyrosine kinase receptor). HEK293T cells (human embryonic kidney cells) transfected with BL-EGFR were incubated with the complex (100 nM), washed, and then treated again with azoc-QPD (20 μM). After 1 h irradiation, clear fluorescence due to the release of QPD was observed (Figure 16). Normal HEK293T cells, which do not express the EGFR, showed no fluorescence after the treatment (Figure 16).167 Similarly, MCF7 breast cancer cells were treated with [Ru(bpy)2(raloxifene-phen)]PF6 (1 μM), incubated 30 min, washed, and treated again with azoc-QPD (5 μM). After 1 h irradiation, clear fluorescence in the nucleus due to the release of QPD was observed. Raloxifene is an estrogen antagonist that binds to the estrogen receptor in MCF7 cells and induces nuclear translocation. Finally, [Ru(bpy)2(O-benzylguaninephen)]PF6 was synthesized and used to label proteins through SNAP-Tag technology. The activation of the azoc-QPD was also successful in cells. This method allowed the targetlocalized labeling through specific ligand interactions.167 3.3. Reduction of Pt(IV) Complexes

Platinum compounds such as cisplatin are known anticancer agents which bind to DNA interfering with transcription. However, platinum(II) compounds are quite reactive and only a small concentration of the drugs will reach their target. On the other hand, platinum(IV) is kinetically less reactive than their PtII analogues. Activation of the PtIV drugs is usually achieved by reduction, accompanied by loss of the axial ligands and release of a PtII compound. As a general approach, PtIV can be activated with cellular reductants (ascorbic acid or GSH) or by light irradiation. Motexafin gadolinium (31) is a texaphyrin gadolinium complex which can catalyze the transfer of electrons from ascorbate to O2, generating ROS.168 Sessler et al. hypothesized that PtIV could act as an electron acceptor instead of O2. Thus, the activation of PtIV prodrugs could be achieved using 31 as a catalyst and ascorbate as reductant.169 For this approach, the PtIV complex should be too hydrophobic to cross the membrane efficiently, and therefore it cannot be reduced to its active PtII form (the inside of the cell is a reducing environment which could lead to the formation of the PtII compound). On the other hand, Sessler et al. has recently demonstrated that compound 31 accumulates preferentially in cancerous tissues (2 μM following a single intravenous dose of 10 μmol/kg).170 The PtIV compound would only be reduced in those areas where 31 accumulated, providing a degree of selectivity. Of special interest in this work is compound [(COOH(CH2)2COO)2Pt(R,R-DACH)(oxalate)] (complex 32, DACH = diaminocyclohexane, Figure 17), in which the two pending carboxylate groups are deprotonated under physiological conditions and, therefore, cannot cross the cell membrane due to the negative charge. The authors have shown that reduction of four different PtIV complexes (including 32) to their corresponding PtII analogues can be achieved successfully using 1 equiv of SODium ascorbate and 0.3 mol equiv of 31 in PBS at pH 7 and 37 °C (Figure 17).169 Although, the presence of O2 does compete with the PtIV compounds as electron acceptors. The absence of

Figure 17. Structure of complex 31, 32, and oxaliplatin (a). Scheme of the reduction of PtIV with 31 (b).

ascorbate, even when using another reductant (GSH), results in no reaction. When A2780 ovarian and A549 lung cancer cells were treated with the PtIV complex 32 and 1 mol equiv ascorbate, toxicity due to reduction of 32 with biological reductants can be observed. However, upon addition of 31 (1 mol equiv, 100 μM), the IC50 values decrease significantly (from 48 to 3.8 μM in A549 cells; from 28.2 to 4.67 μM in A2780).169 Furthermore, the decrease of IC50 values correlate with increasing concentrations of 31.169 Inductively coupled plasma−mass spectrometry (ICP-MS) experiments, also showed that, in the presence of 31, there is 14× times more Pt-DNA adducts than in the absence of gadolinium. Other PtIV complexes were also studied, however, the effect of coincubation with 31 is much lower. The lower enhancement was due to the higher hydrophobicity of the compounds, which allowed higher accumulation in cell of the PtIV which is reduced by cellular reductants inside the cells.169 3.4. Deallylation/Depropargylation Reactions

Many well-known drugs and imaging agents such as rhodamine 110 (RH), doxorubidicin (DOX, anticancer drug), or 5fluoracil (5FU, anticancer drug) contain primary or secondary amino groups. Protecting those functionalities can, in some cases, render the compounds inactive or quench their fluorescent properties. Deprotection of those pro-drugs/ fluorophores has been shown to be a successful strategy for the delivery and amplification of anticancer drugs or fluorophores.36,75 Carbamates are well-known protecting groups for amine functionalities. In particular, allyloxycarbonyl- (Alloc-) and propargyloxycarbonyl- (Proc-) groups are stable to water, air, and against nucleophilic attacks, which makes them suitable to use in biological studies. Similarly, allyl- (Allyl-) and propargyl841


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be used for the modulation of properties in specific drugs and allow further controlled activation.175 The protected DNA binders were also fluorescent, enabling the location of the drug in cells by fluorescence microscopy. In their study, chicken embryo fibroblast (CEF) and Vero (African green monkey kidney) cells were incubated with Alloc-4′,6-diamidino-2-phenylindole (Alloc-DAPI, 2.5 μM) for 30 min and washed. Fluorescence microscopy images show that Alloc-DAPI distributes homogeneously in the cells with no particular preference for DNA. However, subsequent treatment with 2.5 μM of 33 and 100 μM thiophenol induces full deprotection of Alloc-DAPI in 20 min and makes free DAPI shift to nucleus (Figure 19).175

(Pro-) groups can also be used to alkylate amines; as protecting groups, they are also stable in water, air, and against nucleophiles. Other functional groups such as alcohols or carboxylic acids can also be protected using allyl or propargyl moieties. Deprotection of allyl/propargyl- groups is usually performed with palladium catalysts. However, other catalysts such as [Cp*Ru(COD)Cl] (33) or [CpRu(QA)(η3allyl)]+ (35, QA = 2-quinolinecarboxylate) have also been shown to catalyze the deprotection reaction.171−173 3.4.1. Molecular Transition Metal Complexes. In 1999, Mitsudo et al., developed the first RuII catalyst ([Cp*Ru(COD)Cl], 33) capable of cleaving allyl-carbamates and transfer them to sulfur containing nucleophiles (both aliphatic and aromatic) (Figure 18).171 The reaction studied achieved high yields (70−99%) with low catalyst loading (0.05 mol equiv) and under very mild conditions (acetonitrile, RT, 1 h).171

Figure 18. Deprotection of Alloc-NRR′ using [Cp*Ru(COD)Cl] (33) and thiophenol.

Later, Meggers et al. studied the possibility of using 33 and thiophenol to cleave the allyl-carbamate group from Allocmethylaniline under more biologically relevant conditions (37 °C, H2O, and air).174 Interestingly, moderate yields were obtained even in the presence of water or air. It was also demonstrated that no reaction occurred in the absence of thiols.174 Since 2006, the catalytic cleavage of allyl-carbamates using complex 33 under biologically relevant conditions has been studied using different Alloc-protected substrates such as fluorophores,75,174,175 cellular nutrients,82,176 or DNA binders.175 Interestingly, the catalytic reaction occurred even in the presence of high salt concentration, cell growth media, or even in the presence of calf thymus DNA.174−176 In 2006, Meggers et al. reported the first “in cells” experiments using complex 33. In this work, colonies of HeLa cells were treated with 100 μM allyloxycarbonylrhodamine 110 (Alloc-RH), incubated for 30 min and washed before addition of 20 μM complex 33.174 After 15 min of treatment with the RuII complex, a 3.5-fold increase in fluorescence was observed in the cytoplasm due to the release of rhodamine 110 (RH) (10-fold fluorescence increase was observed with the addition of thiophenol to the cell media).174 In addition, the reaction mixture was shown to be nontoxic to the cells at the studied concentrations. In another report, Mascareñas et al. demonstrated that DNA affinity of some DNA binding molecules (ethidium bromide, 4′,6-diamidino-2-phenylindole, or pentamidine) could be greatly reduced by protecting the amine groups with allylcarbamates. However, the DNA binding properties of the molecules could be reactivated by deprotection with complex 33. Thus, it was demonstrated that Alloc protecting groups can

Figure 19. Catalytic deprotection of Alloc-DAPI (a). Fluorescence microscopy image of Vero cells treated with Alloc-DAPI (2.5 μM) and Alloc-DAPI (2.5 μM) + PhSH (100 μM) + 33 (2.5 μM) (b).175 Reproduced by permission of the Royal Society of Chemistry from ref 175. Copyright 2014 Royal Society of Chemistry.

Similarly, CEF cells were incubated with Alloc-ethidium bromide (Alloc-EtBr) for 30 min and washed before treatment with 20 μM of 33 and 100 μM thiophenol. Again, accumulation of the Alloc-EtBr occurs mainly in the cytosol. However, after deprotection, EtBr accumulates in the nucleus.175 These experiments set up the initial steps for the development of less toxic and more controllable DNA-binding drugs. Going a step further, Rotello et al. envisioned a method to selectively switch on/off the activity of [Cp*Ru(COD)Cl] (33) by loading it on 2 nm gold nanoparticles (a nontoxic support) containing a “gate-keeper molecule” (cucurubit[7]uril).177 The gold nanoparticles, capable of penetrating cell membranes, were functionalized with alkene segments (hydrophobic tails where complex 33 can be attached through noncovalent interactions), tetra(ethylenglycol) (provides biocompatibility), and dimethylbenzylammonium (increases solubility and allows the binding of the gate-keeper molecule). After loading with complex 33, the nanoparticles are reacted with cucurubit[7]uril (CB[7], gate-keeper molecule), which interacts with the dimethylbenzylamonium groups, thus blocking the access to the RuIII catalyst that is placed in the nanoparticles (Figure 20).177 The fully prepared nanoparticles 842


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Figure 20. Structure and mechanism of Au-33-CB[7] (a). Confocal microscopy images of HeLa cells incubated with Alloc-rhodamine 110 (AllocRH) and Au nanoparticles, demonstrating RH deprotection inside the cells upon addition of ADA (b).177 Reproduced with permission from ref 177. Copyright 2015 Springer Nature.

Figure 21. Photoactivatable complex [Cp*Ru(η6-pyrene)] (34) for the photocleavage of Alloc-RH under biological conditions.

of the catalytic activity, demonstrating the reversibility of the reaction and providing control over the catalytic process. These gold nanoparticles provide a safe scaffold for the transport of catalysts in a medium which could potentially deactivate the complexes.

(Au-33-CB[7]), were not capable of deprotecting Alloc-RH. However, upon release of CB[7], the catalytic deprotection of carbamates is achieved, yielding the free RH. Release of CB[7] was achieved by addition of 1-adamantylamine (ADA, Figure 20).177 Subsequent addition of CB[7] results in the quenching 843


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originally prepared by Kitamura et al., is a known catalyst for the allylation of alcohols172,173 and has been used for deprotection of allyl-amino acids179 or oligoribonucleotides.180 The complex (25 μM) was capable of conducting allylcarbamate deprotection at pH 7.5 and 25 °C using GSH (5 mM) and as a substrate, Alloc-aminocoumarin (Alloc-AC, a fluorophore also used as an antibiotic) (500 μM), achieving up to 99% conversion in 2 h. In contrast, complex [Cp*Ru(COD)Cl] (33) achieves less than 20% yield and is deactivated after 30 min. The catalytic activity could be further increased by addition of electron donating substituents in the bidentate ligand.74 Compound [CpRu(QA-OMe)(allyl)]+ (36) was significantly more active than compound 35, however, the most active catalyst was found in [CpRu(QANMe2)(allyl)]+ (37, Figure 22).74 The catalytic reactions of 33, 35, 36, and 37 in cells were also studied. In those experiments, HeLa cells were treated with 100 μM Alloc-RH for 30 min, washed, and then incubated with 20 μM of the Ru catalysts.74 In all cases, fluorescence due to the release of RH was observed. The increase of fluorescence followed the same pattern as that observed in noncellular environment (33 < 35 < 36 < 37), with the highest increase in fluorescence (130×) obtained with complex 37 and the lowest increase (8×) with complex 33.74 Deprotection of Alloc-aminoluciferin (Alloc-ALF) using complex 36 was also studied. Upon deprotection, aminoluciferin is released. Aminoluciferin (ALF) interacts with luciferase enzymes, generating light. For kinetic assessment of the catalytic reaction, bioluminescence provides a more accurate measurement than fluorescence and allows for realtime measurement of the catalytic reaction because a signal is only observed after release of ALF.181 To understand whether the reaction occurs inside the cell or in the surrounding area, 4T1 (mouse mammary carcinoma) cells were treated with complex 36, washed several times, and then incubated with Alloc-ALF. Interestingly, poor luminescence was observed when compared with experiments where 4T1 cells were first treated with Alloc-ALF, washed, and then treated with complex 36.181 This suggests that the washing process eliminates complex 36 from the system, demonstrating that the RuII complex is not taken up by the cells, and therefore, the deprotection process occurs extracellularly. This results were corroborated by analysis of the extracellular solution and the washing solutions.181 Mascareñas et al. prepared a new series of compounds containing a derivate of 36 conjugated to a mitochondrial targeting moiety (triphenylphosphonium, TPP). The catalytic cleavage of allyl-carbamates by complex [CpRu(QA-TPP)Cl]+ (38, Figure 22) using Alloc-RH as a substrate was studied, demonstrating much higher activity than the parent compound (36).75 To demonstrate the effect of the TPP, HeLa cells were treated with 25 μM of 36 or 38 (15 min), washed with DMEM-FBS (medium + 5% fetal bovine serum) and PBS and then incubated with Alloc-RH (100 μM). Weak fluorescence was observed when using complex 36 (as previously reported by Wender et al.181).75 In contrast, strong fluorescence was observed with complex 38, suggesting that TPP allows a much higher intracellular retention of the metal complex. In addition, complex 38 was shown to accumulate preferentially in the mitochondria.75 An even higher mitochondrial accumulation was observed when [CpRu(QA-TPP)Cl]+ (38) was exchanged for [CpRu(QA-PDPP)Cl]+ (39, Figure 22) (pyren-diphenylphosphine).75

When HeLa cells were treated with Au-33 (200 nM) for 24 h, washed, and then incubated with Alloc-RH (100 μM) for another 24 h, a strong intracellular fluorescence was observed due to the release of RH. In contrast, no fluorescence was observed in cells treated with Au-33-CB[7]. However, addition of ADA (400 μM) to Au-33-CB[7] treated cells resulted in appearance of fluorescence; this was due to the release of CB[7] and activation of the catalytic deallylation properties of the nanoparticles (Au-33).177 It is particularly interesting that CB[7], ADA, and the nanoparticles (Au-33-CB[7], Au-33) showed no toxicity toward HeLa cells at the concentrations used.177 In 2012, Meggers et al. designed the complex [Cp*Ru(η6pyrene)] (34, Figure 21), which is a photoactivatable version of catalyst 33 and provides more control on the catalytic activation of Alloc-protected molecules.178 Upon irradiation of complex 34 with λ = 330 nm, the pyrene moiety is released and active Cp*Ru fragments formed.178 The activity of complex 34 (100 μM) was then tested for the deprotection of Alloc-RH (0.5 mM) in the presence of different thiols. Only 14% deprotection was achieved after 10 min irradiation (330 nm) at 37 °C in the presence of 5 mM cysteine. However, as in the case of complex 33, the use of thiophenol increased the yield to 93%. The light-activated catalyst was subsequently tested in HeLa cells following the same procedure as with [Cp*Ru(COD)Cl] (33) (100 μM Alloc-RH incubated for 30 min, washed, and followed by addition of 20 μM complex). After 10 min irradiation, a 6-fold increase in fluorescence was observed compared to a 70-fold increase upon addition of thiophenol.178 In an attempt to obtain more catalytically active ruthenium compounds for the uncaging of Alloc-protected substrates, Meggers et al. studied the activity of [CpRu(QA)(Allyl)]+ (35, QA = 2-quinolinecarboxylate, Figure 22). This compound was

Figure 22. Second and third generation of ruthenium half sandwich complexes for the cleavage of allyl-carbamates under biological conditions. 844


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Figure 23. Catalytic deprotection of Proc-RH by Pd(ImPy-TLys) (a). Confocal microscopy image of PC-3 cells treated with 45-Lys(Cy5) and 45Lys(Cy5)/Proc-RH (b).192 Other palladium catalysts used for deallylation reactions in cells (c). Reproduced with permission from ref 192. Copyright 2017 Royal Society of Chemistry. https://creativecommons.org/licenses/by/3.0/.

37, achieving an IC50 of 2 μM, suggesting that nearly 100% of the DOX has been activated.182 In 2007, Koide et al. developed a method for the selective detection of palladium traces in samples containing different drugs.183 In this work, Koide developed a nonfluorescent allyl protected FlCl (m-allyl-FlCl), which can selectively react with Pd0. The palladium was shown to catalytically perform deallylation reactions, deprotecting the m-allyl-FlCl and releasing the fluorescent 2,7-dichloro-6-hydroxy-9-[2(hydroxymethyl)phenyl]-3H-xanthen-3-one (FlCl).183 Unfortunately, PdII had to be reduced initially to Pd0 by addition of PPh 3 , tri-2-furylphosphine or tri-2-furylphosphine/ NaBH4.183,184 PdII had to be detected through a catalytic aromatic Claisen reaction.185 Although the reaction can be performed at various pH, the optimum pH for the best reaction rate/fluorescent signal was 7.186 The same profluorophore was also a very efficient probe for the detection of platinum.186,187 Later, Ahnz et al. prepared a FlCl derivative that was protected with a propargyl group (m-pro-FlCl,).188 This profluorophore (m-pro-FlCl) was shown to be more reactive toward palladium species than m-allyl-FlCl, allowing the depropargylation to occur even when using Pd0, PdII, or PdIV with no additional reductants.188 The Pd compounds used in these studies were Pd(PPh3)4, PdCl2, Pd(OAc)2, Na2PdCl4, or (NH4)2PdCl6. Five-day-old zebrafish were incubated in E3 embryo media containing m-pro-FlCl (20 mM) for 30 min at 28 °C. After washing to remove the m-pro-FlCl from the media, the zebrafish were further incubation in E3 embryo media in the presence of increasing concentrations of PdCl2 for 30 min at 28 °C. Strong fluorescence due to palladium catalyzed depropargylation was observed.188 In a second set of experiments, three-month-old zebrafish were incubated with E3 embryo media containing 500 nM PdCl2 for 24 h, washed, and then treated with E3 embryo media containing m-pro-FlCl (20 mM) for 30 min. After dissection, the organs were

Having developed a catalyst that accumulates mainly in mitochondria. Mascareñas et al. prepared allyl-protected 2,4dinitrophenol (allyl-DNP), which releases 2,4-dinitrophenol (DNP) when deprotected. DNP is a known protonophore, which decreases the mitochondrial membrane potential and switches off ATP production.75 HeLa and Vero cells treated with allyl-DNP, and complex 39 showed rapid damage in the mitochondrial membrane potential. Even more important, while the required dose of DNP necessary to produce damaging effect in the cell is 500 μM, only 150 μM allylDNP was necessary to produce the same effect. This implies that the use of 39 not only allows for the use of an inert prodrug (allyl-DNP) but also allows a decrease on the administered drug.75 By changing the 2-quinolinecarboxylate (QA) bidentate ligand for the more electron donating 8-hydroxyquinolinate (HQ), a further 30-fold increase of catalytic activity was achieved for the deprotection of Alloc-AC.182 Furthermore, addition of electron withdrawing substituents in the HQ ligand achieves even higher catalytic activities. The higher catalytic activity obtained in the uncaging of allyl carbamates was achieved with [CpRu(HQ-COOMe)(Allyl)]+ (40, Figure 22). Complex 40 was able to deprotect Alloc-aminocoumarin (Alloc-AC) even in blood serum (high protein and salt concentrations).182 To demonstrate that activation of prodrugs within the cell might have an enhanced therapeutic effect and lower adverse reactions, both compounds, [CpRu(QA-NMe2)(allyl)]+ (37) and [CpRu(HQ-COOMe)(Allyl)]+ (40), were used for the activation of Alloc-doxorubicin (Alloc-DOX) in HeLa cells and the results compared to those obtained for the treatment of HeLa cells with DOX. Complex 37 was capable of deprotecting Alloc-DOX in cells, however, little effect can be observed when using low Alloc-DOX concentrations.74 The IC50 value (when using 1.0 μM complex) was 15 μM, or 10 times higher than that of doxorubicin itself (IC50 = 1.5 μM). Interestingly, complex 40 displayed a much higher activity than 845


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Pd catalysts are also able to activate propargyl-protected fluorophores in Vero and HeLa cells.191 Similarly to previous studies with Au-33-CB[7], Rotello et al. studied the catalytic activity of “blocked” 2 nm gold nanoparticles loaded with 1,1′-bis(diphenylphosphino)ferrocene-palladium(II)dichloride (44). These nanoparticles (Au-44-CB[7]) were inert. However, upon addition of ADA, CB[7] is released and the remaining fragment (Au-44) was able to deprotect Alloc-RH efficiently inside and outside HeLa cells.177 Dealkylation of 5-fluoro-1-propargyl-uracil (N1-pro-5FU) using Au-44-CB[7]/Au-44 was also studied.177 HeLa cells treated with Au-44 (100 nM) for 24 h, washed, and then treated with N1-pro-5FU (conc from 0 to 1 mM) for a further 24 h showed a gradual decrease in cell survival when increasing the concentration of N1-pro-5FU. On the other hand, cells treated with Au-44-CB[7] showed 100% cell survival even at 1 mM N1-pro-5FU concentration. Addition of ADA (0.05 mM) to the HeLa cells treated with Au-44-CB[7] shows a similar profile to cells treated with Au-44, reaching around 20% cell viability when using 1 mM N1-pro-5FU.177 In an attempt to increase cellular uptake for palladium catalysts, Bradley et al. prepared [Pd(ImPy-TLys)] (45, Figure 23), which contains a trilysine peptide (cell penetrating peptide, which is supposed to facilitate cellular internalization).192 Derivatives containing Lys(Cy5) or Lys(FAM) were also prepared to be able to localize the compounds through fluorescence (Cy5 and FAM are known fluorophores used for staining of cells; Cy5 is cyanine dye, FAM = 5(6)carboxyfluorescein). The complexes (0.1 mol equiv) were able to catalyze the deallylation of propargyloxycarbonyl-rhodamine 110 (Proc-RH) to generate RH under biologically relevant conditions and even in the presence of salts or cell lysate. When PC-3 (prostate adenocarcinoma) cells were treated with complex 45 (30 μM) for 2 h, washed, and then incubated with Proc-RH (50 μM) for 18 h, a strong fluorescence in the cytoplasm and in the nucleus can be observed, proving that the deallylation occurs in cells (Figure 23).192 The complex alone was nontoxic even at 200 μM.192 3.4.2. Metal Nanoparticles. For several years, palladium nanoparticles have been known to catalyze C−C bond forming reactions inside cells such as Suzuki−Miyaura or Sonogashira cross coupling reactions.41 However, in 2011, Bradley et al. showed that some palladium nanoparticles can also be used for the deprotection of allyl carbamates or dealkylation of amines.36,75,193 Polystyrene nanoparticles (5 nm) containing terminal amino groups were charged with fluorescent tags (Cy5.5 or Texas red) and with Pd(OAc)2. Upon reduction, the Pd(OAc)2 generates Pd0 nanoparticles which are encapsulated by the use of the bis-acid chloride of racemic Fmoc-glutamic acid (Figure 24).193 Alloc-RH (500 μM) was treated with the nanoparticles (3.4 μM) (P-Pd-Cy5.5, 46; P-Pd-TR, 47), and deprotection of the rhodamine (RH) was achieved in a low yield. In the presence of glutathione (GSH), deprotection reaches 21% yield in 30 h. The catalytic activity and toxicity of the nanoparticles was also studied in HeLa cell. The compounds were shown to enter the cells, and only low toxicity was observed. The cells were treated with 0.17 μM nanoparticles for 24 h, washed, and then treated with 30 μM Alloc-RH. After 24 h incubation, strong fluorescence due to the release of RH can be observed.193

analyzed, showing Pd mainly accumulates in the brain, eyes, and fin and weakly in the heart and liver.188 Chen et al. demonstrated that other palladium salts and compounds were also capable of catalyzing the deallylation and depropargylation of bis-allyl-/propargyl-oxycarbonyl protected rhodamine 110 (Alloc-RH and Proc-RH), both in PBS and inside HeLa cells.36,75,189 The salts and complexes used for these experiments were Pd(OAc)2, Pd(NO3)2 Na2PdCl4, K2PdCl6, Pd(dba)2 (41, dba = dibenzylidene acetone), or the dimeric (allyl)2Pd2Cl2 (42) (Figure 23). The most active compounds were 41 and 42. The Pd compounds were nontoxic to the cell lines studied at the concentrations used in this study. Particularly interesting, propargyloxycarboxyl groups were found to be significantly more susceptible to cleavage than their allyloxycarbonyl counterparts.189 In a later study, Weissleder et al. studied well-known palladium catalysts capable of performing deallylation or depropargylation reactions in aqueous media and PBS. However, when the reaction with proc/Alloc-RH was performed in more biologically relevant media such as MEM (minimum essential media) or HBSS (Hank’s balanced salt solutions), the efficiency of the palladium salts decreases dramatically.190 It was found that electron deficient ligands are particularly effective at generating the active Pd0 species under biological compatible conditions. The more effective compound for the deprotection reaction was bis[tri(2-furyl)phosphine]palladium(II) dichloride (43).190 Although the compound is an effective catalyst, the solubility and lipophobicity of the compound renders it ineffective for in vivo applications. Aditionally, ligand substitution of the Cl with different biomolecules still occurs. As a consequence of the mentioned drawbacks, the catalysts were then formulated into PLGA (poly(lactic-co-glycolic acid)), and PLGA−PEG polymer nanoparticles (already approved by the FDA) which could increase selectivity, solubility, and stability of the palladium compound. The polymer-Pd particles were shown to be stable in a panel of biologically relevant solvents, furthermore, no significant degradation or aggregation was observed.190 An in vitro study also showed controlled release to up to 50% of the Pd compound over a period of 20 h. More importantly, when tested in HT1080 (fibrosarcoma cells), the compound was able to catalyze the deprotection of Alloc/Proc-RH. The complex was shown to be noncytotoxic at the concentrations used for the in vitro experiment (35 μM). Furthermore, the compounds were also capable of catalyzing Heck reaction in cells using 5diethylamino-2-iodophenyl ester to generate the fluorescent 7diethylaminocoumarin molecule. In their work, tumor accumulation and catalytic activity with aloc/Proc-RH in model mice were also studied, demonstrating that the reaction can be carried out in vivo. In a final experiment, the Pd-NP were used to activate Alloc-doxorubidicine (Alloc-DOX), both in cells and mice models.190 In a recent publication, Mascareñas et al. has investigated the use of Pd-phosphine compounds to overtake the stability issues discussed previously in Weissleder work. In his work, different phosphine-Pd(II) and other commonly used Pd(II) compounds were studied as catalysts for the deprotection of the propargylic groups from different molecules. They demonstrate that although all the Pd(II) salts could perform the catalytic deprotection in water, only the phosphine containing Pd catalysts were able to efficiently catalyze the reactions in cell lysates. Furthermore, they demonstrate that such phosphine846


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anticancer agent that inhibits phosphatidylinositol-3-OH) simultaneously with depropargylation of N1-pro-5FU, decreasing cell viability to 22% after 5 days.194 In a slightly different approach, Unciti-Broceta et al. studied the catalytic activity of palladium nanoparticles which were charged on a polyethylene glycol/polystyrene resin bigger than human cells (150 μm P-Pd, 49) and, therefore, can be used to activate a drug extracellularly.195−199 As expected, the nanoparticles (49) were capable of catalyzing the deprotection of Proc-RH successfully to liberate the fluorescent RH (Figure 25).195,196 To demonstrate the bioorthogonality of the reaction in vivo, zebrafish embryos were injected with 49 after 24 h of fertilization. It is worth noting that the embryos developed normally and no toxicity was observed. When incubating the Zebrafish embryos with 49 and Proc-RH, strong fluorescence can be observed in the area surrounding the nanoparticles. Interestingly, fluorescence in the gastrointestinal system can also be observed, both in the embryos treated with 49 /Proc-RH and in the embryos incubated only with Proc-RH. Thus, Proc-RH deprotection can be achieved by gastrointestinal enzymes.196 When using Alloc-RH instead of Proc-RH, deprotection occurred readily in the absence of the nanoparticles, suggesting that the Allocprotecting group is not bioorthogonal in Zebrafish.195 Activation of anticancer drugs using this approach has also been extensively studied. For example, Unciti-Broceta et al. developed a series of 5-fluoracil derivates protected in the N1 position with an allyl (N1-allyl-5FU), propargyl (N1-pro5FU), or benzyl (N1-bn-5FU) group,196 in the N3 position with a propargyl group (N3-pro-5FU),197 and a N1,N3-doubly protected with propargyl groups (Figure 25).197 Complete deprotection after 24 h (PBS medium, pH 7.4, 37 °C) at the N1 position occurs when using N1-pro-5FU, however, no deprotection occurs with N1-allyl-5FU or N1-bn5FU.196 Deprotection at the N3 position from N3-pro-5FU also occurs, but at a much lower rate, achieving only 85% conversion in 24 h.197 The double protected 5-FU gave only 25% 5-FU and 46% N3 protected.197 In addition, experiments at different pH (6.5−7.5) show that deprotection occurs at a higher rate when using more basic pH. The possibility of deprotecting N3-pro-5FU suggest that propargyl-protected floxuridine (pro-FUdR, anticancer compound derived from 5-fluoracil) could also be carried out in combination with 49 (Figure 25). As expected, complete deprotection was achieved when treating pro-FUdR with 49 at 37 °C in PBS pH 6.5, however, higher deprotection rates can be achieved at pH 7.5.198 Colorectal HT116 cells, sensitive to 5-FU treatment, were used to study the deprotection of propargyl-protected 5-FU. While incubation of the cells with N1-pro-5FU showed very low toxicity compared to the use of 5FU, the combination N1pro-5FU/49 (0.67 mg/mL) displayed similar cytotoxicity. Furthermore, the mechanism of action was also the same than that for 5FU, suggesting that the N1-pro-5FU is, indeed, deprotected inside cells.196 When using the combination N3pro-5FU/49, less than 50% cell viability was observed, however, the slower rate of activation of the drug also reduced the overall impact on the cells.197 The combination of pro-FUdR/49 (0.67 mg/mL) in HT116 cells was also studied, giving similar toxicity to that obtained for the unprotected FUdR.198 However, pro-FUdR alone was up to 560 times less active. In addition, the mechanism of action of pro-FUdR/49 was also the same for FUdR.198

Figure 24. Amine functionalized polystyrene microspheres (5 nm) charged with Pd nanoparticles and glutamic acid-R (a). Transient electron microscopy (TEM) image of the microspheres (b). Confocal microscopy image of HeLa cell treated with 47 + Proc-RH, nucleus stained with Hoechst33342 (c).193 Reproduced with permission from ref 193. Copyright 2011 SpringerNature.

The activation of the antineoplastic drug Amsacrine (Ams) was also studied using this method. HeLa cells were treated with 0.42 μM nanoparticles, washed, and then incubated with 100, 40, or 30 μM Alloc-Amsacrine (Alloc-Ams) for 48 h. Deprotection of the Amsacrine (Ams) occurred, and cell viability decreased to values comparable to those of cells treated with Ams.193 Replacement of the fluorescent moiety for cRGDfE (targeting peptide for αvβ3 receptor, overexpressed in many tumors), yielded P-Pd-cRGDfE (48, Figure 24).194 When treating U87-MG (αvβ3 positive human gioblastoma) cells with the nanoparticles, over 95% cellular uptake was observed in 1 h, however, in MCF-7 (α v β 3 negative human breast adenocarcinoma), cells less than 4% uptake was observed, demonstrating the targeting properties of 48 toward αvβ3 containing cancer cells.194 To study the catalytic allyl carbamate cleavage of the compound, U87-MG (human gioblastoma) cells were incubated with the nanoparticles for 1 h, washed, and then incubated again with propargyloxycarbonyl-cresyl violet (Proc-CrV, nonfluorescent). In this experiment, a 6-fold increase in fluorescence was observed after 18 h due to the deprotection and release of cresyl violet (CrV, fluorophore).194 The possibility of specifically activating anticancer compounds inside cancer cells was studied using 5-fluoro-1propargyl-uracil (N1-pro-5FU), and P-Pd-cRGDfE (48) inside U87-MG cells. The nanoparticles were capable of catalytically depropargylating N1-pro-5FU, releasing the drug in cells and decreasing cell viability to 66% after 5 days.194 Interestingly, the compounds were also capable of catalyzing Suzuki− Miyaura reactions and could be used to synthesize PP-121 (an 847


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Figure 25. Catalytic deprotection of a range of different drugs promoted by Pd nanoparticles.

of action as Gm, suggesting that the toxicity of the Proc/AllocGm with 49 is due to deprotection of the Gm.195 In a last report, a series of vorinostat (SAHA) derivates protected with allyl (allyl-), propargyl (pro-), benzyl (Bn-), or O-1-butyn-3-yl or O-4-propargyloxybenzyl (POB-) groups were prepared. SAHA is a histone deacetylase inhibitor, which is used in cancer therapy.200 SAHA has a strong affinity to complexes with metal ions such as iron or zinc, however, the protected versions have a greatly reduced affinity. In the presence of Pd nanoparticles (48), the propargyl and O-1butyn-3-yl protecting groups are cleaved within 3 h, the allyl group is also cleaved but at a lower rate, and as expected, the benzyl group cannot be cleaved.199 Interestingly, the propargyl group of the O-4-propargyloxybenzyl is also cleaved, leaving behind O-hydroxybenzyl-SAHA (Figure 25). When the deprotection was performed at higher pH (7.2−7.5), the Ohydroxybenzyl-SAHA underwent elimination of the hydroxybenzyl group, releasing SAHA.199 Glioma U87-MG and human lung carcinoma A549 cells were treated with 1 mg/mL palladium nanoparticles and 100 μM of the protected-SAHA. After 5 days, incubation of all the compounds except bn-SAHA showed a significant decrease in cell viability, indicating that the release of SAHA occurs in cells. The mechanism of action of the drug was also studied,

Experiments carried out in HT116 under hypoxia conditions (low O2) gave the same results, suggesting that O2 has no effect on the catalytic reaction.198 The catalytic activation of pro-5-FU or pro-FUdR was also studied using different cell lines, giving similar results. This suggests little effect of the media used for the deprotection reaction when using propargyl-protected drugs.197,198 Unciti-Broceta et al. also prepared a series of gemcitabine (Gm) (an antineoplastic drug used for the treatment of various cancers, particularly pancreatic cancer) derivatives, in which the free NH2 group is protected with allyl- or propargyloxycarbonyl groups (Figure 25). The compounds could then be deprotected during 24 h using 49 in PBS, at pH 7.4 and 37 °C. Interestingly, the Proc-Gm deprotection was much faster and cleaner than that of Alloc-Gm.195 The protected compounds (Proc-Gm and Alloc-Gm) were incubated with pancreatic adenocarcinoma BxPC-3 and MiaPaCa-2 cells. The compounds were stable in cellular conditions, and a 23 times decrease in toxicity was observed when compared to free gemcitabine.195 In contrast, when the cells were treated with 0.67 mg/mL of nanoparticles and various concentrations of Proc-Gm, comparable toxicity with Gm was observed. Using Alloc-Gm instead of Proc-Gm gave lower toxicity due to the less efficient deprotection. The combination Proc/Alloc-Gm with 48 has the same mechanism 848


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Figure 26. Image of zebrafish head injected with gold nanoparticles (50) and incubated in the presence and absence of Proc-RH.202 White arrow shows where the resin with the gold is located. Reproduced with permission from ref 202. Copyright 2017 John Wiley and Sons, used under Creative Commons Attribution License (CC BY).

nutrients; strains: ΔpabA (CGSC strain no. 10483), ΔpabB (CGSC strain no. 9507), and ΔaroC (CGSC strain no. 9865)).82,176 In this work, three Escherichia coli mutant strains incapable of synthesizing p-aminobenzoic acid (PABA, necessary for the production of folic acid)) were grown. The mutant cells were fed using Alloc-PABA (10 μM for 30 min) and washed. Under these conditions, E. coli were not able to grow due to the absence of PABA. Interestingly, upon addition of 33 (2 μM), the mutant E. coli were capable of growing again, suggesting that the catalyst can deprotect Alloc-PABA in cells.176 Addition of external thiols did not increase the cell growth, presumably because the thiols generated by E. coli are sufficient for the deprotection reaction.176 In addition, the growth curve of the cells fed with Alloc-PABA + 33 was similar to those corresponding to the cells fed with PABA, proving that the catalytic deprotection of PABA occurs in cells.176 Balskus et al. also demonstrated that the iron(II) catalyst FeSO4 could be used for the hydroxylation of benzoic acid in the presence of citric acid. This reaction can produce phydroxy-benzoic acid (PHBA), which is a necessary nutrient for cell growth. In their paper, they describe how E. coli mutant cells incapable of generating PHBA were grown and treated with benzoic acid (10 to 200 μM). However, no cell growth was observed. Upon addition of citric acid (0.2 to 4 mM) and FeSO4 (10−200 μM), PHBA was formed, and the cultures containing higher concentrations of substrate and catalyst were rescued.176 3.5.2. Deprotection of Amino Acids and Proteins. Schultz et al. studied the possibility of using complex 33 and thiophenol for the deprotection of Alloc-Lys and Allocmethyllysine in proteins. Using the uncaging strategy, they are capable of synthesizing methyllysine modified proteins using engineered E. coli (BL21(DE3)) that produce proteins containing methyllysine at particular positions.203 Similarly, Chen et al. demonstrated that compounds 41 and 42 were also able to deprotect 82−84% of Alloc-/Proc-Lys using only a 10% loading of catalyst after 8 h reaction under biological conditions (PBS, pH 7.4 and 37 °C).189 However,

confirming release of SAHA and histone deacetylase inhibition.199 In a recent study, 75 μm resin charged with gold nanoparticles (50) was synthesized. Gold, like palladium, has been shown to be able to catalyze a great variety of reactions, particularly those involving alkynes.201 Furthermore, gold has high biocompatibility.201 Catalytic deprotection of Proc-RH in PBS (pH 7.4) at 37 °C in the presence and absence of serum was successfully achieved.202 Addition of thiols to the reaction mixture was shown to deactivate the catalytic depropargylation. However, addition of amine nucleophiles broke the reaction. The effect of GSH, as a major thiol in biological systems, was also studied, showing first an increase in activity and later deactivation of the nanoparticles..202 Catalytic activation of anticancer drugs (pro-FUdR, POBSAHA, and poc-DOX) was studied in A549 cells. Remarkably, while the protected drugs and the nanoparticles (50) were not active by themselves, the combination of both showed potent anticancer activity comparable to that of the unprotected drugs,202 thus strongly demonstrating that deprotection in the cells occurs. In a last set of experiments, zebrafish embryos were injected with a single 50 particle in the optical brain cavity. After injection of the resin, Proc-RH was added to the zebrafish medium and the embryos were imaged after 24 h. A strong fluorescence was observed due to the release of RH (Figure 26). In the absence of the catalyst, no fluorescence was observed, proving the catalytic effect of the gold nanoparticles.202 3.5. Miscellaneous

Even though most research into activation of molecules has focused on the development of bioorthogonal pro-drugs or pro-fluorophores, there are few reports dealing with uncaging of protected natural products or proteins. 3.5.1. Rescue of Auxotroph Colonies. In 2013, Balskus et al. studied the possibility of using complex 33 and its uncaging properties to rescue auxotroph E. coli mutants (Auxotroph = organisms incapable of synthesizing key 849


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3.5.3. Deprotection of Carbohydrates. N-Acetylneuraminic acid (Neu5Ac) is a very common and easy to modify monosaccharide of the sialic acid family (Sias).205 These Sias are a family of sugar units with nine-carbon backbone that are generally found at the cell surface of some bacteria and vertebrate cells. They play an important role in cell differentiation, host−pathogen interaction, and signal transduction.206 More detailed information on the Sias family can be obtained in ref 207. Chen et al. prepared a modified version of Neu5Ac, which contains a propargyloxycarbonyl instead of the acetyl group (Neu5proc).208 Using different palladium species, the Neu5proc can be deprotected, releasing neuraminic acid (Neu).208 It was found that Pd nanoparticles (4−20 nm diameter) gave the highest conversions and reaction rates for the depropargylation reaction. Neu is an unstable monosaccharide that is not usually bioavailable. Still, in cancer cells, Neu could have a dramatic effect because its free amino group can be protonated under physiological conditions, which consequently reduces the negative charges on the cell surface and changes the membrane potential.208 In a set of experiments, Chinese hamster ovaries (CHO) were treated with Neu5proc for 24 h. The cells containing Neu5proc on the cell membrane were then labeled with a fluorophore using click chemistry, thus proving that the propargyl moiety is present in cell surface (vide supra for a detailed discussion of click reactions).208 Upon treatment of the cells with Pd nanoparticles, fluorescence decreases due to the depropargylation catalyzed by the nanoparticles.208 Similar results were obtained in experiments with HeLa cells.208 Importantly, the concentrations of nanoparticles used were shown to be nontoxic for the cells.208 In a separate set of experiments K20 cells (a subclone of the human B lymphoma cell line BJA-B incapable of synthesizing Neu5Ac) were treated with Neu5proc.208 After treatment with palladium nanoparticles, the cell membrane potential was measured.208 A decrease in the membrane potential after treatment with the Pd demonstrates the successful depropargylation and the generation of free amine groups. When Jurkat cells were subjected to the same procedure, the formation of large cell clusters could be observed.208 This method therefore allows for the manipulation of the cell surface potential and clustering.208 3.5.4. Deprotection of Genes. In a recent report, Ward et al. prepared the ruthenium compound [CpRu(QA-biotin)(allyl)]+ (Biot-35).209 This compound contains the Ru(II) moiety capable of performing allyl/propargyl deprotection reactions and the biotin group which allows for the use of streptavidin/biotin technology. To achieve efficient delivery of the Ru catalyst, a biotin was conjugated to a cell penetrating moiety (Biot-CP) and a fluorophore. Both, biotin-CP and Biot-35 were attached to a homotetrameric streptavidin. This approach allows for the protection of the Ru catalyst while increasing cell permeability.209 In the next step, Ward et al. aimed to combine the Ru catalyst approach with a gene switch which is upregulated in the presence of thyroid hormone (T3, triiodothyronine). For this purpose, O-allyl-T3 was prepared and the effect of the Ru complex 35, Biot-35, and streptavidin/Biot-35 in PBS buffer (pH 7.5) at 37 °C was studied. In this work, it was demonstrated that although less active than 35, the Biot-35 compound and the streptavidin/Biot-35 retained the ability of performing propargyl/allyl cleavage.209

the other palladium compounds did not deprotect Alloc-lysine (Alloc-Lys). The same group also prepared a set of tyrosine amino acids which were protected using 1,2-allenyl ether (1,2alle-Tyr), 2,3allenylether (2,3alle-Tyr), allenoate (alleOO-Tyr), and propargylether (pro-Tyr).204 Deprotection of tyrosine could be carried out efficiently using compounds 41, 42, Pd(TAPAd)4 (TAPAd = 1,3,5-triaza-7-phosphaadamantane), and Pd(TPPTS)5 (TPPTS = tris(3-sulfophenyl)phosphine trisodium salt). Interestingly, allenyl protecting groups 1,2-alle- and 2,3alle- were removed more efficiently than pro-. alleOO-Tyr was not studied further because the protecting group was already cleaved under lysate conditions.204 Green fluorescent protein (GFP) containing a protected Lys was then prepared. Yields of 90% and 78% were obtained when deprotecting Proc-lysine in 10 μM GFP-N149-Proc-lys (238 amino acid protein) using 100 μM catalyst 41 and 42, respectively (PBS, pH 7.4, 25 °C for 1 h).189 GFP-N149-protyr and GFP-N149-1,2alle-Tyr were also prepared.204 Deprotection of the tyrosine could be achieved for both modified proteins. However, while 60−90% yields were obtained for the deprotection of 20 μM GFP-N149-1,2alle-Tyr with 100 μM Pd catalyst in PBS buffer at 37 °C for 5 min, only 10−35% yield was obtained with GFP-N149-pro-Tyr under the same conditions.204 To demonstrate that the decaging strategy could be performed with other proteins as well, the experiments were also performed using Taq DNA polymerase (taq-Y671-alle-Tyr). The taq-Y671-alle-Tyr was shown to have no polymerase activity due to the protected tyrosine. Treatment of taq-Y671-alle-Tyr with 10 μM Pd complexes successfully deprotected the tyrosine residue and restored the polymerase activity to the protein.204 Modified HeLa cells that express GFP-Y4o-Proc-Lys (GFP protein containing Proc-Lys in Y40) were treated with 10 μM of complex 42 to obtain the deprotected GFP.189 The unreacted protected protein was then reacted with azide-Cy3 (fluorescent probe) through click chemistry and analyzed by gel electrophoresis. An efficiency of 31% in the deprotection reaction was observed (3 h reaction time), showing that the reaction can be conducted in cells.189 The same method was then applied on HeLa cells producing OspF-K134-Proc-Lys (analogue of OspF, a phosphothreonin lyase, and epigenic modulator by dephosphorylation). After treatment with compound 42 (10 μM), 28% deprotection yield was obtained. This method shows that lysine deprotection by palladium compounds can be used to activate lysine-dependent enzymes inside host cells.189 Modified HEK293T cells expressing GFP-Y40-1,2alle-Tyr were incubated for 2 h with 20 μM Pd catalyst. It was found that 42 (51% yield) and 41 (37% yield) could effectively deprotect the GFP-Y40-1,2alle-Tyr,204 and the difference was shown to be due to their different cellular uptake.204 Similarly, cells expressing Src kinase-Y416-1,2alle-Tyr (the protection inhibits the kinase activity of the protein) were treated with 20 μM of 41 or 42 for 3 h. Deprotection of the tyrosine residue occurred successfully, and the kinase activity was restored.204 Finally, also the anthrax lethal factor (LF, key toxin effector of Bacillus anthracis which exhibits protease activity on MEK3 family kinase) was modified to contain a 1,2-alle-Tyr in Y278 (necessary for activity). After deprotection of the 1,2-alle-Tyr with 20 μM of 41 or 42 for 3 h, the protease activity was again restored.204 850


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Figure 27. Suzuki−Miyaura reaction catalyzed by compound 52 (a). Coupling of B-Fl to OmpC in the cell membrane (b).235 Fluorescence of genetically modified E. coli after reaction with B-Fl in the presence and absence of complex 52 (c).235 Chemical structure of ADHP (d). Reproduced with permission from ref 235. Copyright 2009 American Chemical Society.

cine (HPG), propargyloxycarbonyl-lysine (Proc-Lys), p-iodophenylalanine (IPhe), or azido-homoalanine (Aha), is necessary.79,80,210,215 Various reviews on methods for insertion of such unnatural amino acids have been published.211,215,229 In general, cross-coupling reactions have also been used extensively for the synthesis of many active molecules and drugs.230 It is not surprising that with the development of more biologically tolerant reaction conditions for such reactions, the in situ preparation of active compounds from bioorthogonal components, allowing for the delivery of active drugs with minimal side reactions, have instigated considerable interest.193,194,231 In this section, we will discuss the use of metal-catalyzed cross-coupling reactions which can be used for the modification of proteins under biologically relevant conditions and in cells or for the in situ preparation of active agents.76,77,81,216

Finally, HEK-293T cells transfected with the T3-responsive gene switch were treated with streptavidin/(Biot-35)x (biotCP)y (homotetrameric streptavidin can accept up to four biotin groups, therefore x + y = 4), incubated for 1 h, washed, and incubated again in the presence of O-allyl-T3 for 24 h. The release of T3 triggers the activation of histone deacylation, and the process can then be monitored by the conversion of furimazine into a luminescent compound. It was shown that the catalytic reaction occurs inside the HEK cells and the best catalyst was identified as streptavidin/(Biot-35)3 (BiotCP)1.209

4. IN SITU LABELING OF PROTEINS AND SYNTHESIS OF MOLECULES For many years, there has been an interest in studying and understanding the structure, function, and mechanism of proteins, enzymes, antibodies, and other cellular components.210,211 As a consequence, a great variety of studies in protein modifications and biomolecule labeling have been performed. Labeling and modification of biomolecules in vitro has been successfully achieved by S−H insertions in cysteine212 or alkylation of amino acids such as lysine213 or tyrosine.214 Such transformations will not be covered in this work because those reactions cannot be carried out inside cells due to the harsh conditions of low pH required.41,79,80,215,216 Recently, however, there have been an increasing number of studies to develop more cell-friendly labeling methods. Crosscoupling reactions, such as Suzuki−Miyaura,217−219 Sonogashira,220,221 Heck,222−224 copper catalyzed azide−alkyne cycloadditions, or Staudinger,225 tetrazine,226,227 or oxime228 ligations, have attracted much attention due to the stability of the newly formed covalent bonds. However, in many cases, addition of unnatural amino acids, such as homopropargylgly-

4.1. Suzuki−Miyaura Reactions

Suzuki−Miyaura cross-coupling reactions are a very versatile and robust method for the synthesis of organic compounds.218 It is, therefore, not surprising that there has been a great interest in developing a Suzuki-type reaction which could be used in aqueous media or even inside cells. Particularly, the irreversible linkage of small functional molecules to amino acids, peptides, and proteins could lead to multiple applications in biochemistry and biomedicine.217,219,232−234 In 2009, Davis et al. envisioned an efficient phosphine-free palladium catalyst (Pd(OAc)2(ADHP)2 (compound 52, ADHP = 2-amino-4,6-dihydropyrimidine), which could catalyze the Suzuki−Miyaura reaction in aqueous media using unnatural amino acids containing halogen-phenyl moieties (Figure 27).235 The reaction gave nearly quantitative yields when reacting Boc-4-iodo-L-phenylalanine (Boc-IPhe) 851


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Figure 28. NHC ligands used for Suzuki−Miyaura reactions (a). Fluorescence microscopy images of HeLa cells pretreated with n-succinimidyl-piodobenzoate and then treated with biotin−boronic acid in the presence and absence of catalyst 53 and finally with streptavidin−Alexa488 (b).232 * Hoechst blue dye for staining DNA. Chemical structure of ADHP (d). Reproduced with permission from ref 232. Copyright 2014 American Chemical Society.

yields,238 and later, different oligodeoxynucleotides were also coupled with a variety of sensitive and useful functional groups through halogenated pyrimidine bases.239 However, we will not discuss these experiments here in more detail because no in vivo applications were investigated. Four different E. coli colonies (strain JW2203-1) were modified to encode outer membrane protein C containing IPhe (IPhe-OmpC) in four different possitions. The protein is a bacterial membrane protein involved with different infectious processes. When the modified E. coli cells were treated with 0.35 mM compound 52, 1.6 mM boronic acid−fluorescein derivate (B-Fl) for 1 h at 37 °C, and pH 8, an increase of the fluorescence could be observed due to the Suzuki−Miyaura reaction (Figure 27).240 Increasing concentrations of boronic acid or Pd catalyst gave rise to higher activities until a threshold was reached (2 mM boronic acid, 345 μM catalyst). In addition, the reaction yield decreased with lower temperatures, being completely inhibited at room temperature. The reagents were also shown to be nontoxic at the concentrations used.240 In another set of experiments, genetically modified E. coli expressing OmpC containing IPhe in Y232 position were treated with galactose, maltose, or glucose−boronic acid derivates (3.5 mM) and compound 52 (0.5 mM) for 1 h. Efficient binding was observed.241 Interaction of the glycanselective binding proteins Lens culinaris agglutinin (binds

and phenyl boronic acid using only 1% catalyst loading (buffer pH 8 at 37 °C for 4 h).235 Similarly, nearly quantitative yields could be achieved with other iodobenzene derivates and Boc4-bromo-L-phenylalanine.235 Modified subtilisin Bacillus lentus (SBL) containing a piodobenzyl cysteine was also studied as a substrate for the Suzuki−Miyaura coupling with phenyl boronic acid (500 mol equiv) using 50 equiv of compound 52 at 37 °C after 30 min, giving a nearly quantitative yield.235 Interestingly, nearly quantitative yields were also obtained when using different boronic acids, including lipidated ones.235 A number of other proteins such as the genetically encoded His-maltose binding protein containing IPhe were also studied, showing the versatility of the Suzuki coupling. However, analysis by mass spectrometry showed that there is nonspecific binding of the Pd to the protein, which could be reduced using 3mercaptopropionic acid as a scavenger.236 In further studies, the flexibility of this catalyst in Suzuki− Miyaura reaction with different biologically relevant molecules and boronic acid substrates was demonstrated. For example, Liu et al. used (Pd(OAc)2(ADHP)2 (52) to catalyze the ligation of the dye 3-(dansylamino)phenylboronic acid with the genetically modified z domain protein containing IPhe (prepared using pylRS-tRNA).237 Davis et al. achieved the labeling of amino acids and proteins with the radionuclide 18F under really mild conditions, albeit with relatively low 852


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selectively to mannose) and Griffonia simplicifolia lectin I (binds to galactose) was then studied, displaying high specificity of recognition.241 Chen et al. synthesized a series of PdII complexes bearing water-soluble NHC-type bidentate ligands.232 Their catalytic activity was first studied for Suzuki−Miyaura cross coupling reactions by reacting Boc-IPhe and 3-(hydroxymethyl)phenylboronic acid (1.5 mol equiv) with 1% loading of catalyst in phosphate buffer at 37 °C for 2 h. The more active compound was Pd(OAc)2(MTAPI)2 (53, MTAPI = 1-methyl3-[3-(tetramethylamonio)propyl)]-imidazolium)) with a 77% yield. More bulky NHC ligands reduce slightly the catalytic activity of the complexes. Similar results could be obtained using biotin−boronic acid (B-Biot).232 Bovine serum albumin (BSA) was reacted with piodophenyl)methyl p-nitrophenyl carbonate in order to attach a iodophenyl moiety to the lysine residues of the protein (Ipoc-Lys-BSA). Suzuki−Miyaura reaction between Ipoc-LysBSA (7 μM) and B-Biot or B-Fl using compound 53 in phosphate buffer (pH 8) at 37 °C showed robust crosscoupling within 1 h. Biotin-Lys-BSA can be reacted with fluorescent Alexa488. Similarly, the reaction was also successful using n-succinimidyl p-iodobenzoacetyl-lysozyme.232 Going one step further, Chen et al. treated HeLa cells with n-succinimidyl-p-iodobenzoate (60 μM) for 40 min in order to attach an iodobenzene moiety on the cell membrane.232 After washing, the cells were treated with 200 μM of B-Biot and 80 μM of catalyst 53 for 1 h. Subsequently, cells were treated with fluorescent streptavidin−Alexa488, which binds tightly to biotin. Strong green fluorescence can, then, be observed on the surface of the cells, indicating that the Suzuki coupling of biotin to the cell was successful (Figure 28).232 In the absence of the catalyst or the B-Biot, no fluorescence can be observed. Important, an MTT assay performed in HeLa cells treated with the palladium catalyst showed no significant toxicity up to 200 μM.232 The catalytic activity of palladium compounds and nanoparticles toward Suzuki−Miyaura reactions has been intensively studied for the labeling of different biomolecules. However, in a completely different approach, Bradley et al. studied the possibility of catalyzing the cross coupling reaction for the in situ synthesis of either fluorophores or drugs. In his initial reports, nonfluorescent monotriflate-fluorescein (MtFl) was reacted with triphenyl[4-[[4-(4,4,5,5-tetramethyl-1,3,2dioxaborolan-2-yl)phenyl]amino]butyl]-phosphonium (Bphosphonium) in the presence of Pd nanoparticles 47 or PPd-MitoTracker deep red. Upon coupling, the final molecule showed a strong fluorescence.193 In-cell synthesis of the dye was also studied. To this end, HeLa cells were incubated with Pd nanoparticles for 24 h, washed, and then treated with 20 μM MtFl and 20 μM B-phosphonium. After 48 h, green fluorescence can be observed in confocal microscopy due to the Suzuki−Miyaura reaction product.193 Similarly, nonfluorescent bis-iodo-1,3,5,7,8-pentamethyl-bodipy was reacted with either 2-thienyl or 4-phenyl boronic acid to generate fluorescent bodipy analogues. The reaction was achieved successfully intracellularly on prostate adenocarcinoma PC-3 cells.231 The Pd nanoparticles were also applied to the in situ synthesis of PP-121 (anaplastic tyroid carcinoma tumor growth inhibitor).231 The compound can be synthesized with a 62% yield by Suzuki coupling between 1-cyclopentyl-3-iodo-1Hpyrozolo[3,4-b]pyridine-4-amine (IPyr) and 3-(4,4,5,5-tetra-

methyl-1,3,2-dioxaboralan-2-yl)-1H-pyrrolo[2,3-b]pyridine (Bpyr) in the presence of the Pd nanoparticles. The cross coupling reaction was then tested in the extracellular media of PC-3 cells by incubating 10 μM BPyr and 2 μM IPyr with 5 μmol Pd nanoparticles for 5 days. Cell viability decreases by 50% due to the formation of PP-121. Interestingly, none of the individual components showed significant toxicity in PC-3 cells under the concentrations used.231 4.2. Sonogashira Reactions

Sonogashira-type reactions to label amino acids, peptides, or proteins has been previously reported in the literature. The reaction proceeds using palladium catalysts and a CuI additive even in aqueous solutions. However, the reaction conditions used are often not biologically compatible. In addition, the use of CuI species in biological systems is not adequate due to the toxicity associated. In 2011, Lin et al. developed a method which allowed the labeling of proteins via Sonogashira coupling using a palladium catalyst but no Cu(I) cocatalyst.220 In his work, HPG-bngly (homopropargylglycine-benzyloxycarbonylglycine) was reacted with IFl (2.4 equiv) compounds using Pd(OAc)2DADHP (compound 54, 30% loading, DADHP = 2-dimethylamino-4,6dihydroxypyridine), and ascorbate (1 mM) in phosphate buffer at 37 °C to obtain the cross coupling product with a 91% yield. The reaction was equally successful with a variety of aryl iodide ligands, with only slightly lower yields when using electron deficient aryl iodides.220 In a later set of experiments, Lin et al. also studied the effect of the microenvironment in the Sonogashira reaction.221 A series of peptides containing HPG in the middle of the sequence were prepared and then used as substrates for the Sonogashira reaction. Interestingly, it was found that while some sequences gave a nearly quantitative yield, other sequences gave almost no product.221 Addition of a specific peptide sequence containing HPG to the C-terminus of ubiquitin (Ubi-PepHPG) indeed helped to increase the Sonogashira yields compared to ubiquitin containing only a terminal HPG (Ubi-HPG) when using compound 54.221 The increased reaction rate is due to the effect of the surrounding amino acids which can sequester the aryl iodide as if it was a protein binding pocket. Furthermore, while some bulky aryl iodides gave low yields for the reaction with Ubi-HPG,220 the sequence of amino acids on UbiPepHPG had low selectivity toward the type of aryl iodide, allowing the use of a great variety of substituted compounds (including bulky or fluorescent aryl iodide units).221 Thus, it was demonstrated that the surroundings of the alkyne group exert an important effect on the reaction. More details on the surrounding microenvironment can be found in the ref 221. Propargyloxycarbonyl-Boc-Lys-Gly-Bn (Proc-LysG) and 3butyn-1-yloxycarbonyl-Boc-Lys-Gly-Bn (Butoc-LysG) were also prepared as alkyne carriers to replace HPG.242 Their reaction with a variety of aryl iodides was also tested in the presence of 54 (30% loading) and ascorbate in biologically relevant conditions. Sonogashira product was obtained with both substrates; however, only Butoc-LysG gave a nearly quantitative yield.242 To prove that the Sonogashira cross coupling reaction could be achieved inside living organisms, E. coli (M15A) expressing HPG-Ubi was treated with 1 mM compound 53, 100 μM fluorescein iodide (IFl), and 5 mM ascorbate for 4 h. After washing, the cells displayed strong green fluorescence due to the labeling with fluorescein.220 853


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Figure 29. Chemical structure of DADHP (a). Sonogashira reaction between Ipo-Biot and HPG (b) or Butoc-Lys (c). Confocal micrographs of HEK 293 cells expressing EGFR-EGFP containing Pep(HPG) (d) or Butoc-Lys(128) (e) treated with 100 μM Ipo-Biot and in the presence or absence of compound 54 (100 μM). Reproduced with permission from ref 242. Copyright 2015 American Chemical Society.

Mammalian HEK293 human embryonic kidney cells expressing peptide Pep(HPG)-EGFR-EGFP (Pep(HPG) = GRYFS(HPG)PRPSR = specific sequence containing HPG inserted in between residues Ala-21 and Ser-22 of EGFREGFP (EGFR-EGFP = epidermal growth factor receptor with EGFP fused) were treated with the palladium catalyst (50 μM) and p-iodophenyl-biotin (PI-biot, 50 μM). After 30 min, the biotinylation was monitored using Alexa568 conjugated streptavidin. Red fluorescence demonstrated that Sonogashira reaction occurs on the surface of mammalian cells (Figure 29). Controls with Pep(HPG)-EGFR-EGFP and no Pd catalyst or expressing Pep(Met)-EGFR-EGFP showed no fluorescence.221 Similarly, E. coli (DH10B) expressing myoglobin-butoc-Lys was also treated with IFl and the palladium catalyst 54, the yield of fluorescent labeling obtained was 67% using the same conditions than before. The results obtained with myoglobinbutoc-Lys were compared to those obtained when the Sonogashira reaction was performed in cell lysate, showing similar results.242

HEK293 cells expressing Butoc-Lys(128)-EGFR-EGFP (located in cell membrane) were treated with 100 μM 54 and p-iodophenyloxy-biotin (Ipo-Biot) for 30 min. The cells were subsequently treated with fluorescent streptavidin− Alexa568 for 20 min and washed with PBS. Confocal laser scanning microscopy showed high fluorescence indicating that the Sonogashira incorporation of biotin was successful (Figure 29).242 HEK293 human embryonic kidney cells, CHO Chinese hamster ovary cells, and HeLa cells were used to test the toxicity of the Pd complex and the Ipo-Biot. Gratifyingly, no significant toxicity at the experimental concentrations was observed.242 Chen et al. developed a ligand-free method for the Sonogashira reaction. In this work, a series of Pd salts and complexes (including 54, Pd(NO3)2 and Na2PdCl4) or nanoparticles were used to attach the labeling fluorophore iodophenyl-PEG-Fluor 525 (Ipo-PEG-FL525), 4-pentyn-1yloxycarbonyl-PEG-Fluor 525 (Pentc-PEG-FL525), 4-pentyn1-yloxycarbonyl-PEG-biotin (Pentc-PEG-biot), or iodopheny854


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Figure 30. Complexes for olefin metathesis (a). Ring closing metathesis scheme of 5-hydroxy-2-vinylphenyl acrylate (HVPA) (b). E. coli cells treated with 5-hydroxy-2-vinylphenyl acrylate and SAVperi-biotin-55.2 or SAVperi (c). Reproduced with permission from ref 255. Copyright 2016 SpringerNature.

loxy-PEG-Fluor 525 (ipo-PEG-biot) into green fluorescent protein (GFP) containing either p-iodo-phenyloxycarbonyl lysine (Ipoc-Lys-GFP) or pentyn-1-yloxycarbonyl lysine (Pentc-Lys-GFP) as unnatural amino acid. Proc-Lys was not used as palladium compounds will induce depropargylation. Similarly to the case of Suzuki−Miyaura reactions,243 the PEG linker enhanced the reaction efficiency by stabilizing catalytic intermediates. The Sonogashira reaction with all Pd compounds was successful, however, the best yields were obtained when using Pd(NO3)2 as a catalyst. Interestingly, labeling using Ipoc-Lys-GFP was significatively less successful than when Pentc-Lys-GFP was used. This may be due to the fact that Pd reacts first with the I-phenyl moiety, and accessibility of IpocLys-GFP might be sterically hindered.244 When using Pd(NO3)2 as a catalyst, concentrations of the catalyst as low as only 2 mol equiv could be used to couple ipoPEG-FL525 (10 mol equiv) and Pentc-Lys-GFP, giving a 95% yield in the presence of 1 mM ascorbate. In addition, the Sonogashira reaction with Pd(NO3)2 could be performed at 25 °C. Interestingly, the ligand-free catalyst Pd(NO3)2 allows the use of much milder conditions than Pd catalysts containing organic ligands such as 53 (reaction at 37 °C, 1 mM catalyst, and 50 mol equiv Pentc-Lys-GFP).244 Under the conditions mentioned, there was no denaturalization of GFP under any conditions. However, when studying a more sensitive protein such as Pentc-luciferase, catalysts such as 53 denaturized the protein, while Pd(NO3)2 had no denaturizing effect.244 E. coli (strains: EPEC and EHEC) cells expressing PentcLys-GFP were incubated with Pd(NO3)2 (200 μM) and IpoPEG-FL525 (200 μM) for 1 h at 25 °C. Confocal fluorescence microscopy confirmed that Sonogashira reactions can occur inside E. coli cells. Similarly, it was also demonstrated that

labeling of Pentc-OspF (bacterial type 3 secretion effector) inside Shigella cells can also be performed successfully.244 4.3. Metathesis Reactions

Olefin metathesis has been established as a useful, versatile, and tolerant to functional groups synthetic tool for the formation of CC bonds. Since 1950, many applications and a vast number of catalysts for such reactions have been developed, including some which can tolerate the presence of air and water.245−247 It is, therefore, not surprising that the possibility of using such reactions for applications in chemical biology has been explored as well. Some compounds such as the first- and second-generation Hoveyda−Grubbs catalyst (HG catalyst, 55) were shown to successfully catalyze metathesis under mild or biologically compatible conditions, including unnatural amino acids,248 peptide synthesis,249,250 or proteins.251,252 Despite the fact that HG catalyst has shown some tolerance for functional groups in proteins, concentrations of the catalyst required for protein modification are still relatively high.251 Accordingly, Ward and Hilvert coordinated a HG catalyst with a protein scaffold which could increase the solubility of the compound and, more importantly, act as a protecting environment for the catalyst.253,254 In the first approach, the Methanocaldococcus jannaschii small heat shock protein 16.5 containing a single free cysteine (MjHSP) was coordinated to a modified version of the HG catalyst.253 Compound MjHSP55.1 (4% loading) was then used to catalyze the metathesis of N,N′-diallyl-4-toluenesulfonamide (5 mM) to give N-(ptoluene-sulfonyl)-3-pyrroline under biological conditions, however, the reaction gave low yields (3%). More acidic conditions (pH 2) increase the reaction yield (25%).253 855


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(strain: M15MA[pQE-60/OmpC]).261 In this work, the outer membrane protein C (OmpC) was engineered to contain six azidohomoalanine (Aha) residues (N3-OmpC) exposed to the outside of the cell. E. coli cells containing the N3-OmpC and OmpC were then treated with CuSO4 (100 μM), tris(carboxethyl)phosphine (TCEP, 200 μM), biotin-PEG-alkyne (50 μM), and Tris-[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA, 200 μM). Labeling of the membranes of E. coli containing N3-OmpC was successful, while E. coli expressing OmpC were not affected by the CuAAC reaction. Optimal conditions of the reaction were studied.261 The direct use of CuI (CuBr) instead of the CuII system (CuSO4/TCEP) gave rise to a much higher catalytic activity for the CuAACs.262 Unfortunately, CuI is easily oxidized in biological systems and large copper concentrations are necessary. In addition, the use of copper agents is quite damaging for the cell components due to the formation of ROS when CuI is regenerated by reduction with ascorbate.259,262,263 Finn et al. synthesized the ligand Tris-[(1-hydroxypropyl1H-1,2,3-triazol-4-yl)methyl]amine (THPTA), which showed better solubility than TBTA, and, in combination with the Cu/ ascorbate system, was capable of promoting CuAAC while avoiding oxidation of amino acids and protein damage as a consequence of H2O2 production.264 Interestingly, in the absence of the THPTA ligand, 65% oxidation of histidine was observed after 20 h to CuSO4 (0.5 mM)/ascorbate (2 mM) catalysis. However, when using THPTA (5 mol equiv compared to Cu)/CuSO4 (0.5 mM)/ascorbate (2 mM) catalysis, only 5% oxidation after 20 h was observed. Interestingly, the THPTA was consumed in the process, suggesting that it is used as a reductant for H2O2.264 In addition, using aminoguanidine (amG) is capable of capturing dehydroascorbate (which may otherwise react with proteins). The applicability of this method was then studied in HeLa and CHO mammalian cells. In the experiment, the cells were first incubated with 50 μM N-azidoacetylmanosamine, which is taken up and incorporated in the cell surface-located sialylated glycans. After washing, the cells were further treated for 5 min with a preprepared mixture (10 min reaction before cell addition) of CuSO4/THPTA (5:1), aminoguanidine (1 mM), alkyne-modified rhodamine (alk-Rh), and ascorbic acid (2.5 mM). An incubation period of 5 min was previously shown to give good yields with little or no toxicity, while extended incubation times increased the toxicity greatly. Cell labeling was observed by confocal microscopy.265 In 2009, Brotherton et al. found that when using an azido substrate which can bind to the CuI center as a chelating ligand, the reaction rate was greatly enhanced.264 This was ascribed to the higher electrophilicity on the azide (for a suggested mechanism see ref 264). Following this approach, Ting et al. studied the catalytic activity of CuAACs between 7ethynylcoumarin (fluorescent, 20 μM) and 6-azidomethyl-3pyridinecarboxylic acid (PyN3, 40 μM) or 4-azidomethyl benzoic acid (PhN3, 40 μM) using CuSO4 (10 μM)/ascorbate (4 mM) as a catalyst and at pH 7.4 (phosphate buffer) and 25 °C. While no reaction was detected after 30 min when using PhN3 as a substrate, 38% yield was observed using PyN3. This increase in yield and reaction rate was attributed to the chelating effect of the substrate with the copper and was confirmed with different substrates.266 Using higher concentration of catalyst (CuSO4, 40 or 100 μM) allows for a 100% yield for PyN3 within 10−15 min, while no reaction occurs with PhN3. Addition of a ligand such as THPTA (4 mM)

In a second approach, Ward et al. prepared a series of biotinHG catalysts which have a strong affinity toward avidin and streptavidin (SAV).254 Compound biotin-55.1 (0,73 mM) was able to catalyze the metathesis of N,N′-diallyl-4-toluenesulfonamide (15,21 mM) with a 77% yield when the reaction was run in DMSO (16%)/H2O for 16 h at pH 7 and 40 °C.254 Addition of streptavidin forms SAV-biotin-55.1 and gives a 99% when using 2.5 ppm of compound 56 (25 ppm copper). However, no reaction occurred in the absence of MONPs. Those results were also obtained using a variety of other substrates.267 NCI-H460 human nonsmall cell lung carcinoma and MDAMB-231 human breast cancer cells were treated with 0.5 μM compound 56 for 1 h. After washing the cells, they were treated with 2 mM ascorbate, 100 μM 3-azido-7-hydroxycoumarin (N3-CU, nonfluorescent), and 100 μM p-ethynylanisole. Strong fluorescence to the CuAAC reaction in cells could be observed by confocal microscopy, while no fluorescence was observed in the absence of 56. Similar results were obtained when using 2-picolylazide and 7-ethynylcoumarin. In addition, cytotoxic assays demonstrated acceptable biocompatibility.267 In a final set of experiments, E. coli were treated with 56, washed, and then treated with ascorbate/4-azido-N-[3-(4,5dihydo-1H-imidazol)phenyl]-benzamide (N3-benzymide)/N[3-(4,5-dihydo-1H-imidazol)phenyl]-4-ethynylbenzamide (Albenzymide). Both N3-benzymide and Al-benzymide have no antibacterial activity toward E. coli (MIC > 300 μg/mL), however, the cycloaditition product shows a MIC value of 11 μg/mL.267 Interestingly, strong inhibition of bacterial growth was observed, suggesting the formation of bisamidine inside cells.267 In 2016, Bradley et al. developed a 160 μm resin bead (ECu-NPs, 57) containing copper nanoparticles (51 nm, 0.37 μmol Cu/mg resin),268 and the nanoparticles were prepared as described for 50 and 49. The nanoparticles (0.3 mg) were capable of performing CuAACs between N3-CU (50 μM) and

4.5. Catalysis by Gold(III) Complexes

Gold compounds in the oxidation state + III are known to react with alkene and alkyne groups, making the multiple bonds reactive toward a variety of nucleophiles. Various reports have been published, which use AuIII ions to promote cyclization and fluorescence activation in different fluorophores containing alkyne groups.282−285 Those studies aimed to selectively detecting free gold ions in cells and were also studied in different cell lines with successful results.282−286 Tanaka et al. prepared a AuIII cyclometalated compound conjugated to coumarin which was capable of activating propargyl esters. Upon coordination of the propargyl ester to the AuIII compound, amination with amino groups in the 857


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we included some examples of Suzuki−Miyaura and Sonogashira-type PEGylation reactions. Davis et al. studied the possibility of using Suzuki-type reactions for the controlled PEGylation of amino acids, peptides, and proteins under biological conditions. In this work, they studied the use of PdII as a catalyst in the absence of any ligand (K2PdCl4) or in combination with 2-amino-4,6dihydropyrimidine (Pd(OAc)2(ADHP)2, compound 52), 2(N,N-dimethyl)-amino-4,6-dihydroxypyrimidine (Pd(OAc)2(DADHP)2, compound 54), 1,1-dimethyl guanidine (Pd(OAc)2(DMGu)2), and 1,1,3,3-tetramethyl guanidine (Pd(OAc)2(TMGu)2).243 Suzuki coupling between boc-IPhe and PEG20K-PBA (20 kDa monomethoxy PEG phenylboronic acid, 1.5 mol equiv) was achieved in moderate to high yields when using 20% loading in all the cases except for compound 52. Similarly, using a shorter PEG chain gave similar results with only 5% catalyst. The reaction was not dependent on the concentration of catalyst but on the concentration of the PEG ligand. When the reaction was performed using only the 4-(Nmethylamino-carbonyl)-phenylboronic acid (with no PEG chain), the Suzuki reaction using K2PdCl4 was greatly hampered, but good yields were again achieved by addition of external PEG, demonstrating that PEG works as a stabilizing additive for the PdII.243 Genetically modified Bacillus lentus subtilisin (SBL) and allβ-helix protein 275−276 Nostoc punctiforme containing IPhe were prepared and used as model proteins for PEGylation. Similarly to the case of boc-IPhe, PEGylation was achieved with moderate to good yields in all the cases except when using catalyst 52. The higher reaction yields were obtained when using K2PdCl4.243 Chen et al. also demonstrated that 20 mol equiv of compound 53 can catalyze the reaction between 500 mM PEG2K-PBA and n-succinimidyl-p-iodobenzoate-lysozyme in phosphate buffer at 37 °C. Within 2 h, a 90% yield of PEGylation could be observed. Lin et al. also studied the PEGylation of proteins, however, using Pd catalysts for a Sonogashira reaction. In this work, genetically modified myoglobin containing Proc-Lys or ButocLys was prepared and reacted for 30 min with m-PEG-linked phenyl iodide (100 mol equiv) using compound 54 (100 mol. equiv) and ascorbate under biologically relevant conditions. The reaction was successful, giving 73% PEGylated product for the Butoc-containing myoglobin, and only 30% for the Proccontaining myoglobin.

surface proteins occurred. When the propargyl ester was attached to fluorescent probes, this method could be used as in situ labeling.287 Tanaka et al. also demonstrated that N-glycoalbumin can be modified to exhibit organ-selective accumulation.287 For example, α(2-6)-disialoglycoalbumin is retained within the liver, while galactose−glycoalbumin is directed to the intestines.287 Because coumarin has a strong affinity for albumin, the gold catalyst can be selectively directed to a particular organ, which can later be labeled using profluorescent probes. In this paper, BALB/cAJcl-nu/nu mice were injected with the Au−albumin compound. After 30 min, Cy7.5-propargylester was also injected. Noninvasive fluorescence imaging was then performed over 2 h, and organtargeted labeling was observed.287 In a recent work, Mascareñas et al. have reported several phosphine containing gold(I) and gold(III) complexes which are capable of promoting carbocycloaditions with derivatives of coumarin precursors under biologically relevant conditions. The only exception tested was [AuCl(PPh3)] (58), although the poor catalytic activity was suspected to be due to low solubility. It is particularly interesting that compound [AuCl(PTA)] (59) showed very good yields and selectivity. Additionally, the reaction was shown to still work in the presence of different cellular components such as carbohydrates, amino acids, or GSH, although poisoning could be observed when using an excess of some of these components such as GSH. The reaction was also proven to work in the presence of PBS, Dulbecco’s Modified Eagle’s Medium (DMEM) or bovine serum albumin. HeLa cells were then incubated for 30 min with the gold compound (50 μM), followed by washing and further incubation with the coumarin (100 μM) precursor for 6 h. A strong fluorescence due to the reaction could be observed. In addition, experiments with LysoTraker showed that the compounds accumulate in the lysosomes. These experiments were performed only with compounds displaying no toxicity at working concentrations. In a final set of experiments, Mascareñas et al. attempted to perform Au catalysis while also performing deprotection reactions using complex 38. In these experiments, HeLa cells were treated with the gold compound (50 μM) and complex 38 (25 μM) for 30 min and then washed and incubated again with the corresponding substrates (derivative of coumarine precursor and a fluorophore protected with a propargyl group). After 6 h incubation, strong fluorescence to the reaction with Au can be observed. Similarly, strong fluorescence to the deprotection reaction can be observed. This demonstrates that both reactions could be performed in parallel.288

5. PERSPECTIVE In this review, we summarize work that has been published on catalytic metallodrugs as defined in the Introduction. The field already brings fascinating new insights into biologically relevant questions by merging two separate but otherwise firmly established fields in transition metal chemistry, namely catalysis and metal-based drugs. It is interesting to note that intracellular metal-catalyzed transformations on biomolecules (beyond ROS production and photodynamic therapy, which are out of the scope of this review) were already occasionally used to influence biological systems before the year 2000, e.g., hydrogenation of membrane lipids in bacteria and algae.47,90−97 On the other hand, the field has really taken off with the work by Meggers and co-workers to catalytically deprotect fluorogenic probes intracellularly, and by the seminal work of Sadler and co-workers, who discovered methods to shift important intracellular equilibria such as GSH/GSSG or

4.6. Miscellaneous Reactions

4.6.1. PEGylation of Proteins. PEGylation of proteins is a common approach used for the development of protein-drugs because it allows the improvement of stability and biodistribution of the protein. PEGylation allows for the reduction of clearance rate in the body while providing a steric shield against immune system recognition and other enzymes.242,243,289 Despite the fact that PEGylation has not been performed inside cells, some cross-coupling reactions under biological conditions have been studied. Because of their relevance in the development of Pd catalysts that can be used under biologically relevant conditions and later inside cells, here, 858


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NAD+/NADH by transition metal catalysis.106,114−116,123,125−127,75,174 In a second stage, indirect effects of mingling with important intracellular processes have also been identified, for example, an increased (or gained back) sensitivity of cancer cells against commonplace therapeutic drugs.136 As described in this review, quite a number of different biological questions were already tackled in diverse biological systems. However, it does still seem as if the field is largely driven by chemists and their chemistry in the sense that for a given type of system/catalyst/transformation that can be used or performed, a suitable biological application is chosen as proof of principle. Surely, at this point, the field would benefit from more biological input to identify challenging biological questions, where a catalytic transformation might provide insights, and the required chemical system would have to be designed and optimized toward that purpose. When compiling this review, we were quite impressed by the diversity of chemical systems that were already employed, ranging from molecular transition metal complexes to nanoparticles and multilayer systems. As in many other fields of life, we feel that diversity here is really beneficial, as it allows one to choose the optimal system for any one given situation to be studied. One other aspect of note lies in the fact that in traditional transition metal catalysis, a large part of the time is usually spent to carefully optimize the reaction conditions (solvent, temperature, maybe pressure), so as to achieve up to 100% conversion and highest enantiomeric purity where needed. In a biological environment, the situation is different in that the catalyst (and where possible the substrates) really are the only parameters that may be optimized. Conditions are fixed to whatever the biological system dictates, typically with temperatures slightly above room temperature, an aqueous environment, and (usually) the presence of air and hence oxidizing conditions. Hence, there is a real art in choosing the type of reaction/catalyst system that is still functional under those challenging boundary conditions. While reactions such as Sonogashira coupling and the famous Cu-catalyzed azide− alkyne cycloaddition reaction (CuAAC) are obvious candidates and were the first to be described in the literature, we strongly feel that this is the area where new and clever chemistry will likely bring the most exciting advances in the future, at least from a chemistry perspective. It will be exciting to see which reactions can be adapted to the required conditions next or even how the challenge of a biological environment as a conditio sine qua non (a condition that has inevitably to be met) will inspire new chemistry and totally new transformations. In this sense, the field of catalytic metallodrugs is well set to bring together chemists and colleagues from biology and biomedical sciences to explore new problems and engage in new interactions and collaborations across disciplinary boundaries.

Biographies Joan Josep Soldevila Berreda received his B.S.c in Chemistry at Universitat de Barcelona (Spain) in 2010. From 2010 to 2014 he obtained his Ph.D. in Inorganic and Medicinal Chemistry from Warwick University (U.K.) under the supervision of Prof. Peter J. Sadler. He obtained an IAS early career fellowship (2014−2015) from Warwick University to conduct postdoctoral research at Prof. Peter J. Sadler’s laboratory. In 2015, he joined Prof. Nils Metzler-Nolte’s group at Ruhr University Bochum (Germany) as an Alexander von Humboldt postdoctoral fellow. Presently, he is a postdoctoral research assistant at Dr. Nicolas Barry group at the University of Bradford. Nils Metzler-Nolte obtained his Ph.D. from LMU Munich (Germany) in 1994, did a postdoc with Prof. M. L. H. Green at Oxford (U.K.), and started his independent research on bioorganometallic chemistry at the Max-Planck-Institut für Strahlenchemie (nowadays MPI for Chemical Energy Conversion) in Mülheim, Germany. He was appointed Associate Professor for Pharmaceutical and Bioinorganic Chemistry at the University of Heidelberg in 2000, and Full Professor of Bioinorganic Chemistry at the Ruhr University Bochum in 2006. He served as Dean of the University-wide graduate school from 2009−2012 and was Vice President for Early Career Researchers and International Affairs of his university between 2010 and 2012. Nils was Speaker of the DFG-funded Research Unit “Biological Function of Organometallic Compounds”, and Council Member of the Society of Biological Inorganic Chemistry. His work was recognized by several fellowships and awards, the most recent being the Julius von Haast award of the Royal Society of New Zealand. Together with Kathy Franz, he was Chair of the Gordon Research Conference “Metals in Medicine” in 2016. Nils serves on the international advisory board of several journals (including inter alia Chemical Science, Organometallics, and Journal of Biological and Inorganic Chemistry) and is an Associate Editor for Dalton Transactions. With research interests in medicinal organometallic chemistry and functional metal bioconjugates, his group is running a full program from inorganic synthesis to cell biology.

ACKNOWLEDGMENTS J.J.S.B. is grateful for a fellowship from the Alexander-vonHumboldt Foundation. N.M.-N. is grateful to the many members of his research group over the years, not only for their excellent work but also for an outstanding working atmosphere that made “work” feel like a passion, not like a duty, on every single day. A big THANK YOU! to the group! And despite the many distractions from outside our research that I (and sometimes all of you) have to cope with, keep up the spirit and your good work! SUMMARY OF COMPLEXES DISCUSSED 1 = [Pd(QS)2] 2 = [Cp*Rh(bipy)Cl]+ 3 = [(hmb)Ru(en)Cl]+ 4 = [(p-cym)Ru(TsDPEN)Cl] 5 = [(bn)Ru(TfEn)Cl] 6 = [(p-cym)Ru(TsEn)Cl] 7 = [(Cp*)Rh(TfEn)Cl] 8 = [(CpxPhPh)Rh(en)Cl]+ 9 = [(p-cym)Os(TsDPEN)Cl] 10 = [(hmb)Ru(bpm)Cl]+ 11 = [(ind)Ru(bpm)Cl]+ 12 = [(Cp*)Ir(phen)Cl]2+ 13 = [CpXPhIr(phen)Cl]2+ 14 = [CpXPhIr(PhPy)py]2+

AUTHOR INFORMATION Corresponding Author

*Phone: +49-2343228152. E-mail: [email protected]. Website: www.chemie.rub.de/ac1. ORCID

Nils Metzler-Nolte: 0000-0001-8111-9959 Notes

The authors declare no competing financial interest. 859


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15 = [(Cp*)Ir(bipy)Cl]2+ 16 = [(Cp*)Ir(6,6′-dihydroxybipy)Cl]2+ 17 = [(Cp*)Ir(N-phenyl-2-pyrimidinecarboxamidate)Cl] 18 = [(Cp*)Ir(4-(1H-pyrazol-1-yl-κN2)benzoic acid-κC3)H2O)]2SO4 19 = [(p-cym)Os(ImPy-NMe2)Cl]+ 20 = [Cp*Ir(biot-p-L)Cl] 21 = [(bip)Ru(azopy-NMe2)I]+ 22 = [(p-cym)Ru(o-bqdi)I]+ 23 = [(CpxPhPh)Ir(bpy)SH]+ 24 = [(CpxPhPh)Ir(phen)SH]+ 25 = [(p-cym)2Ru2(μ-SC6H4-p-CH3)3]+ 26 = [(Cp*)2Rh2(μ-SC6H4-p-CH3)3]+ 27 = [(p-cym)6Ru6(dhbq)3(tpt)2]6+ 28 = [(p-cym)6Ru6(dnbq)3(tpt)2]6+ 29 = [Fe(TPP)Cl] 30 = Ru(bpy)3Cl2 31 = motexafin gadolinium 32 = [(COOH(CH2)2COO−)2Pt(DACH)(oxalate)] 33 = [Cp*Ru(COD)Cl] 34 = [Cp*Ru(η6-pyrene)] 35 = [Cp*Ru(QA)(η3-allyl)]+ 36 = [CpRu(QA-OMe)(allyl)]+ 37 = [Cp*Ru(QA-NMe2)(η3-allyl)]+ 38 = [CpRu(QA-TPP)Cl]+ 39 = [CpRu(QA-PDPP)Cl]+ 40 = [Cp*Ru(HQ-COOMe)(η3-allyl)]+ 41 = Pd(dba)2 42 = allyl2Pd2Cl2 43 = bis[tri(2-furyl)phosphine]palladium(II) dichloride 44 = [(1,1′-bis(diphenylphosphino)ferrocene)palladium(II) dichloride] 45 = [Pd(ImPy-TLys)] 46 = P-Pd-Cys5.5 47 = P-Pd-TR 48 = P-Pd-cRGDfE 49 = 150 μm P-Pd 50 = 75 μm P-Au 51 = [Pd(QS)2] 52 = (Pd(OAc)2(ADHP)2 53 = Pd(OAc)2(MTAPI)2 54 = Pd(OAc)2(DADHP)2 55 = second generation Hoveyda−Grubbs catalyst 56 = Cu-MONPs 57 = E-Cu-NPs 58 = [AuCl(PPh3)] 59 = [AuCl(PTA)]

MCF7 = human breast cancer cells MDA-MB-231 = human breast cancer MiaPaCa = human pancreatic carcinoma MRC5 = healthy lung fibroblast NIH-3T3 = mouse embryon fibroblast NCI-H460 = human nonsmall cell lung carcinoma PC3 = human prostate cancer cells SKOV-3 = human ovarian adenocarcinoma U87-MG = human gioblastoma cells Vero = African green monkey kidney cells Bacteria

E. coli = Escherichia coli, Gram-negative bacteria Shigella = Gram-negative bacteria

SUMMARY OF SUBSTRATES 5FU = fluoracil (anticancer drug) N1-pro-5FU = 5-fluoro-1-propargyl-uracil (no anticancer activity) N1-allyl-5FU = 5-fluoro-1-allyl-uracil (no anticancer activity) N1-bn-5FU = 5-fluoro-1-benzyl-uracil (no anticancer activity) N3-pro-5FU = 5-Fluoro-3-propargyl-uracil (no anticancer activity) Aha = azydohomoalanine (unnatural amino acid) ALF = aminoluciferin (bioluminescent upon interaction with luciferase enzymes) Alloc-ALF = allyloxycarbonyl-aminoluciferin (no interaction with luciferase enzymes) Ams = amsacrine (anticancer drug) Alloc-ams = allyloxycarbonyl-amsacrine (no anticancer activity) B-phosphonium = triphenyl[4-[[4-(4,4,5,5-tetramethyl1,3,2-dioxaborolan-2-yl)phenyl]amino]butyl]-phosphonium benzymide Al-benzymide = N-[3-(4,5-dihydo-1H-imidazol)phenyl]-4etynylbenzamide (no antibacterial activity) biotin = vitamin B7, strong affinity for streptavidin and avidin Ipo-Biot = p-iodophenyloxy-biotin B-Biot = biotin bound to boronic acid Bodipy = 1,3,5,7-tetramethyl-8-phenyl-4,4-difluoroboradiazaindacene (fluorophore) Bodipy-CHO = (T-4)-[4-[(3,5-dimethyl-1H-pyrrol-2-ylκN)(3,5-dimethyl-2H-pyrrol-2-ylidene-κN)methyl]benzaldehydato]difluoroboron (nonfluorescent) Bodipy-OH = (T-4)-[4-[(3,5-dimethyl-1H-pyrrol-2-yl-κN)(3,5-dimethyl-2H-pyrrol-2-ylidene-κN)methyl]benzenemethanolato]difluoroboron (fluorophore) Bpyr = 3-(4,4,5,5-tetramethyl-1,3,2-dioxaboralan-2-yl)-1Hpyrrolo[2,3-b]pyridine (component of PP-21) BSA = bovine serum albumin CA4 = combrestatin A4 (tubulin polimerization inhibitor, anticancer activity) CU = coumarin (precursor for some pharmaceutics, fluorescent) ACU = aminocoumarin (antibiotic and fluorophore) Alloc-ACU = allyloxycarbonyl-aminocoumarin (nonfluorescent) N3-CU = 3-azido-7-hydroxycoumarin (low fluorescence quenched by azide) CrV = cresyl violet (fluorophore)

CELL LINES Mammalian Cells

4T1 = mouse mammary carcinoma A2780 = human ovarian cancer cells A549 = human alveolar epithelial adenocarcinoma, lung cancer cells BJAB-K20 = subclone of human B-lymphoma cells BxPC3 = human pancreatic adenocarcinoma CEF = chicken embryo fibroblast CHO = Chinese hamster ovarian cells HeLa = human cervical cancer cells (Henrietta Lacks) HEK293 = human embryionic kidney cells HT116 = human colon carcinoma Jurkat cells = leukemic T-cell lymphoblast 860


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Neu5proc = propargyloxicarbonylneuraminic acid OspC = outer surface protein C OspF = phosphothreonin lyase PABA = p-aminobenzoic acid Alloc-PABA = allyloxycarbonyl-p-aminobenzoic acid PHBA = p-hydroxy-benzoic acid Phe = phenylalanine IPhe = p-iodophenylalanine (unnatural amino acid) PEG20K-PBA = 20 kDA monomethoxy PEG phenylboronic acid PEG2K-PBA = 2 kDA monomehoxy PEG phenylboronic acid PP-121 = inhibitor of phosphatidylinositol-3-OH (anticancer agent) pro-NTPP = [6-oxo-6-(2-propyn-1-ylamino)hexyl]triphenylphosphonium QPD = quinazolinone (fluorophore) Azoc-QPD = azidophenyloxycarbonyl-quinazolinone (nonfluorescent) RH = rhodamine 110 (fluorophore) N3-RH = azido-rhodamine 110 (nonfluorescent) Alloc-RH = allyloxycarbonyl-rhodamine 110 (nonfluorescent) Proc-RH = propargyloxycarbonyl-rhodamine 110 (nonfluorescent) Src kinase = proto-oncogene tyrosine-protein kinase Src Tyr = tyrosine 1,2alle-tyr = 1,2-allenyl tyrosine ether (unnatural amino acid) 2,3alle-tyr = 2,3-allenyl tyrosine ether (unnatural amino acid) alleOO-tyr = tyrosine allenoate (unnatural amino acid) pro-tyr = propargyl tyrosine ether (unnatural amino acid) SAHA = vorinostat (anticancer agent) POB-SAHA = O-4-propargyloxybenzyl vorinostat (no anticancer activity) bn-SAHA = benzyl-vorinostat (no anticancer activity) Ubi = ubiquitin Umb = umbelliferone (fluorophore)

Proc-CrV = propargyloxycarbonyl-cresyl violet (nonfluorescent) DAPI = 4′,6-diamidino-2-phenylindole (DNA intercalator, fluorescent) Alloc-DAPI = allyloxycarbonyl-4′,6-diamidino-2-phenylindole (no DNA affinity, fluorescent) DNP = 2,4-dinitrophenol (antiseptic and pesticide; in cells inhibits synthesis of ATP) allyl-DNP = allyl-2,4-dinitrophenol (antiseptic and pesticide; in cells inhibits synthesis of ATP) DOX = doxorubidicin (anticancer drug) Alloc-DOX = allyloxycarbonyl-doxorubidicin (no anticancer activity) EMBA = 5-ethynyl-2-methoxybenzenamine EtBr = ethidium bromide (DNA intercalator, fluorescent) Alloc-EtBr = allyloxycarbonyl-ethidium bromide (no DNA affinity, fluorescent) EGFR-EGFP = epidermal growth factor receptor Fl = fluorescein (fluorophore) IFl = 5′-iodo-fluorescein; fluorescein derivate (fluorophore) MtFl = 2-[3-oxo-6-[[(trifluoromethyl)sulfonyl]oxy]-3Hxanthen-9-yl]benzoic acid, fluorescein derivate (nonfluorescent) FlCl = 2,7-dichloro-6-hydroxy-9-[2-(hydroxymethyl)phenyl]-3H-xanthen-3-one, fluorescein derivate (fluorophore) m-allyl-FlCl = 2,7-dichloro-9-[2-(hydroxymethyl)phenyl]-6(2-propen-1-yloxy)-3H-xanthen-3-one, allylether protected FlCl (nonfluorescent) m-pro-FlCl = 2,7-dichloro-9-[2-(hydroxymethyl)phenyl]-6(2-propyn-1-yloxy)-3H-xanthen-3-one (nonfluorescent) FUdR = floxuridine (anticancer drug derived from 5fluoracyl) Pro-FUdR = (N3)-propargyl-floxuridine (no anticancer activity) GFP = green fluorescent protein (fluorogenic protein) Gm = gemcitabine (anticancer agent) Alloc-Gm = allyloxycarbonyl-gemcitabine (no anticancer activity) Proc-Gm = propargyloxycarbonyl-gemcitabine (no anticancer activity) GSH = glutathione GSSG = oxidized glutathione HPG = homopropargylglycine (unnatural amino acid) HVPA = 5-hydroxy-2-vinylphenyl acrylate (nonfluorescent) IPyr = 1-cyclopentyl-3-iodo-1H-pyrozolo[3,4-b]pyridine-4amine (component of PP-121) Lys = lysine Alloc-Lys = allyloxycarbonyl-lysine (unnatural amino acid) Butoc-Lys = 3-butyn-1-yloxycarbonyl-lysine (unnatural amino acid) Ipoc-Lys = p-iodo-phenyloxycarbonyl-lysine (unnatural amino acid) Pentc-Lys = 4-pentyn-1-yloxycarbonyl-lysine (unnatural amino acid) LF = anthrax lethal factor N3-benzymide = 4-azido-N-[3-(4,5-dihydo-1H-imidazol)phenyl]-benzamide (no antibacterial activity) N3-TMOB = 5-azido-1,2,3-trimethyloxybenzene NAD+ = nicotinamide adenine dinucleotide NADH = nicotinamide adenine dinucleotide reduced Neu = neuamic acid Neu5Ac = acetilneuraminic acid

PROTECTING GROUPS 1,2-alle = 1,2-alleny 2,3-alle = 1,2-alleny AlleOO = allenoate allyl = allyl Alloc = allyloxycarbonyl Azoc = azidophenyloxycarbonyl Boc = tert-butyloxycyrbonyl Bn = benzyl Butoc = 3-butyn-1-yloxycarbonyl Ipo = p-iodo-phenyl Ipoc = p-iodo-phenyloxycarbonyl Mt = monotriflate 861


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N3 = azido Pentc = 4-pentyn-1-yleoxycarbonyl Pro = propargyl Proc = 2-propargyloxycarbonyl POB = O-4-propargyloxybenzyl

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