Bioorthogonal Azide–Thioalkyne Cycloaddition Catalyzed by Photoactivatable Ruthenium(II) Complexes

Click Reactions Bioorthogonal Azide–Thioalkyne Cycloaddition Catalyzed by Photoactivatable Ruthenium(II) Complexes Alejandro Guti8rrez-Gonz#lez, Paolo Destito,Jos8R. Couceiro,Cibran P8rez-Gonz#lez, Fernando Llpez,* and Jos8L. MascareÇas* Abstract: Tailored ruthenium sandwich complexes bearing photoresponsive arene ligands can efficiently promote azide– thioalkyne cycloaddition (RuAtAC) when irradiated with UV light. The reactions can be performed in abioorthogonal manner in aqueous mixtures containing biological components.The strategy can also be applied for the selective modification of biopolymers,such as DNAorpeptides. Importantly,this ruthenium-based technology and the standard copper-catalyzed azide–alkyne cycloaddition (CuAAC) proved to be compatible and mutually orthogonal. Introduction Bioorthogonal reactions,byenabling the covalent modification of specific reactants or biomolecular targets in complex biological environments,have brought aparadigm shift on the potential of chemistry for interrogating or/and altering biology.[1,2] Within the “toolbox” of bioorthogonal reactions,those that are catalyzed by transition metals are especially attractive,owing to their intrinsic metal-dependent characteristics,and the possibility of tuning the reactivity by adjusting the characteristics of the catalyst.[3] However, progress in this field has been slow,ingreat part because of the notion that transition metal reagents are incompatible with aqueous and biological milieu, and that they can be easily inactivated by biological components.Moreover,while there has been an increasing number of reports on bioorthogonal metal-catalyzed reactions,they usually present low catalytic efficiencies,especially under the diluted conditions usually required for biological applications.[3,4] Among all transition-metal-mediated bioorthogonal reactions,there is one that stands out, namely,the copper(I)- promoted azide–alkyne cycloaddition (CuAAC).[5,6] Thereaction engages organic azides and alkynes,which are ideal chemical entities in terms of biological orthogonality,and tends to exhibit very good rates.However,this transformation still presents important limitations such as its low compatibility with thiols,its restriction to terminal alkynes or the side reactivity and toxicity of copper (I) ions in biological contexts.[7] Furthermore,toreach efficient conversions under typically diluted conditions,the reactive copper(I) species need to be generated in situ using excess amounts of acopper(II) source and sodium ascorbate,areductant which is not innocent in biological contexts (Figure 1).[8] Therefore, there is aclear need to discover new,robust and aqueouscompatible metal-catalyzed annulations as alternatives to the CuAAC.[9,10] In this context, in 2017, we reported the first examples of aruthenium-catalyzed azide–alkyne cycloaddition that takes place in aqueous and in biologically relevant milieu.[11] The method makes key use of thioalkynes as reaction partners, and of the commercially available complex [Cp*RuCl(COD)] (Ru1)ascatalytic reagent (Figure 1). Following our report, other groups developed alternative conditions to achieve Figure 1. Metal-catalyzed azide–(thio)alkyne cycloaddition reactions. Advantages are marked with blue squares, and limitations with red. [*] A. Guti8rrez-Gonz#lez, Dr.P.Destito, Dr.J.R.Couceiro, Dr.C.P8rez-Gonz#lez, Dr.F.Lkpez, Prof. J. L. MascareÇas Centro Singular de InvestigaciknenQu&mica Biolkxica eMateriais Moleculares (CiQUS) and Departamento de Qu&mica Org#nica Universidade de Santiago de Compostela 15782 Santiago de Compostela (Spain) E-mail:j[email protected] Dr.F.Lkpez MisiknBiolkgica de Galicia Consejo Superior de Investigaciones Cient&ficas (CSIC) 36080 Pontevedra (Spain) E-mail:f[email protected] Supportinginformation and the ORCID identification number(s) for the author(s) of this article can be found under: https://doi.org/10.1002/anie.202103645. T2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH. This is an open access article under the terms of the Creative Commons Attribution License, which permitsuse, distribution and reproduction in any medium, provided the original work is properly cited. A ngewandte Chemie Research Articles How to cite: Angew.Chem. Int. Ed. 2021,60,16059–16066 International Edition: doi.org/10.1002/anie.202103645 German Edition: doi.org/10.1002/ange.202103645 16059Angew.Chem. Int.Ed. 2021,60,16059–16066 T2021 TheAuthors.AngewandteChemieInternational Edition publishedbyWiley-VCH GmbH related cycloadditions in water, using nickel,[12] rhodium[13] or iridium catalysts,[14] albeit only the latter has been shown to operate in biological mixtures. Importantly,all these new metal-catalyzed azide–alkyne cycloaddition reactions,including our ruthenium-catalyzed process,have demonstrated effectivity only when the concentration of reagents is in the mid to high millimolar range. This represents an important limitation in terms of developing biological applications,which usually require very diluted samples.Anadditional challenge in this field has to do with the possibility of controlling the reactivity using external stimuli, as this could open new opportunities for biological regulation. Herein, we demonstrate that cationic RuII complexes such as [Cp*Ru(MeCN)3]PF6(Ru2)are excellent precatalysts to perform efficient, formal cycloadditions between thioalkynes and azides in aqueous media. Contrary to [Cp*RuCl(COD)] (Ru1), this cationic complex (Ru2)isvery effective under diluted micromolar conditions,even in PBS and biologically complex media, such as DMEM, or HeLa cells lysates.More importantly,wealso show that the azide–thioalkyne cycloaddition can be catalyzed by [Cp*RuIIarene] sandwich complexes,[15] provided that they are photoactivated by ashorttime irradiation with aLED lamp at 365 nm (Figure 1). The possibility of controlling the generation of the catalytically active RuII species with light opens interesting perspectives in optobiology.[16] Finally,wealso demonstrate that this technology is fully orthogonal with the CuAACand, moreover,it can be used for the chemoselective modifications of small peptides and ssDNAs,for instance,for the introduction of fluorogenic tags. Results and Discussion Our first experiments were carried out with the anthracenyl azide 1a and the thioalkyne 2a,because the resulting triazole product (3aa)isfluorescent and, thus,the reaction can be readily monitored. In consonance with previous observations,the neutral RuII complex [Cp*RuCl(COD)] (Ru1)was significantly more efficient than [Cp*Ru- (MeCN)3]PF6(Ru2), when the reaction was carried out under anhydrous conditions in CH2Cl2(75 mM).[17] Specifically,the reaction gave a99%yield of the product after 0.5 hwith Ru1 (Table 1, entry 1), but just a15% yield with Ru2,after 1h (30%yield after 6h,entry 2). Several control experiments and careful analysis by NMR and ESI-MS allowed to discover that the poorer performance of Ru2 was likely due to the formation of secondary ruthenium-containing products.In particular,wecould identify Ru2’’,which results from an unprecedented ruthenium-promoted trimerization of thioalkynes,aprocess that generates achelating dithiofulvene ligand (Figure 2).[18] Theperformance of Ru2 in the RuAtAC could be partially improved using Et3NCl as additive (5 mol%), probably by favoring the in situ formation of aneutral Cp*–ruthenium(II) chloride species (entry 3), which might hamper the thioalkyne-to-fulvene trimerization.[19] Remarkably,when the reaction catalyzed by Ru2 was carried out in water, in the presence of this chloride source, the cycloadduct 3aa was obtained in 99%yield, after only 0.5 h(entry 4). We later found that the use of Et3NCl is not needed, as the reaction provided the same yield without any additive (entry 5). Most likely,the higher activity of Ru2 in water is partially related to the formation of active RuII aquo or oxo derivatives,which favor the desired annulation over alternative pathways.[20] Indeed, we have detected by ESI-MS several ruthenium-oxygenated species in the aqueous solutions of Ru2.[21] Importantly,using these conditions,wecould promote the annulation of avariety of azides and thioalkynes (Scheme 1). Thioalkynes bearing an ethyl group at the sulfur atom were particularly reactive,but other alkyl groups like benzyl (2c), isopropyl (2d)oraromatic substituents (2b)are also tolerated, providing in all cases the expected products in good yields.The other substituent of the thioalkyne can also be modified without compromising the yields of the desired triazoles. Ru2 proved to be much more selective than the previously described catalyst Ru1 with respect to the type of alkyne partner used. Indeed, Ru1 promotes the reaction of regular alkynes lacking the thioether,such as the 2fand 2g,togive the corresponding triazoles in moderate to good yields (3bf, 3bg,Scheme 1). However, the cationic reagent Ru2 failed to induce any conversion with these alkynes,even after 7hat rt Table 1: Viability of the RuAtAC with the RuII cationic complex Ru2.[a] Entry [Ru] (%) Solvent t[h] Yield [%][b] 1Ru1 (5) CH2Cl20.5 99 2Ru2 (5) CH2Cl2115(30)[c,d] 3[e] Ru2 (5) CH2Cl2128(72)[c] 4[e] Ru2 (5) H2O0.5 99 5Ru2 (5) H2O0.5 4 [a] Reaction conditions: 2a (150 mmol), 1a (75 mmol), solvent (1 mL) and the ruthenium catalyst (5 mol%) were added to avial under air,and the mixture stirred for the indicated time. [b] Yield determined by 1HNMR spectroscopy of the reaction crude mixture using 1,3,5- trimethoxybenzene as an internalstandard. [c] The yield after 6his indicated in parenthesis. [d] The complex Ru2’’was detected in the reaction mixture (NMR and ESI-MS).[18] [e] The Ru complex Ru2 and Et4NCl (5 mol%) were premixed in the corresponding solvent for 5min. Note:Reaction mixtures in water can be considered as suspensions rather than solutions. Figure 2. Structure and X-ray crystallographic analysis of Ru2’’(specific hydrogen atoms and the counterion (PF6@)are omitted for clarity). A ngewandte Chemie Research Articles 16060 www.angewandte.org T2021 TheAuthors.AngewandteChemieInternational Edition publishedbyWiley-VCH GmbH Angew.Chem. Int.Ed. 2021,60,16059–16066 (Scheme 1). Therefore, Ru2 not only allows to carry out the annulation in aqueous media in an efficient manner,but also introduces alevel of chemoselectivity that was previously unattainable with Ru1,allowing to fully distinguish thioalkynes from alkynes (both mono- and disubstituted). Considering this chemoselectivity,wenext explored the orthogonality of the RuAtACand the CuAAC. When amixture of azide 1b (1 equiv.) and alkynes 2a and 2h (2 equiv.ofeach one) was treated under standard CuAAC conditions [that is,CuSO4·5H2O(5mol%) and sodium ascorbate (NaAsc,10mol%)] the triazole 3bh,resulting from the reaction of the terminal alkyne 1h was exclusively formed in high yield (Table 2, entry 1). If,after this reaction has been completed (2 h), we add asecond equivalent of the azide,and the catalyst Ru2 (5 mol%), the thioether containing adduct 3bais formed in agood 78%yield (entry 2). Even more relevant, when the initial mixture of the azide 1b and alkynes 2a and 2h is first treated with Ru2,only the sulfurcontaining triazole 3ba is observed (79%yield, entry 3), whereas the subsequent addition of 1a and the Cu catalyst leads to 3bhin 95%yield (entry 4). Furthermore,when azide 1b was mixed with both 2a and 2h (1 equiv.each) in the presence of both Cu and Ru catalysts (5 mol%ofeach one), an almost equimolar mixture of 3ba and 3bh is obtained, in an excellent yield (94%yield, entry 5). Overall, these results confirm astriking chemoselectivity and mutual orthogonality between both methods and indicate that both cycloadditions share similar kinetic profiles under these conditions.This mutual orthogonality promises relevant applications,such as for the dual tagging of biomolecules.[22] In view of the excellent performance of Ru2,weexplored its behavior under more diluted conditions.Gratifyingly,the annulation between 1cand 2acan be efficiently carried using azide concentrations of 1mM(99%yield), and even at 250 mM, without significant deterioration in the yield (86% yield).[23] At this point we questioned the possibility of engineering ruthenium derivatives that could be activated using external stimuli, owing to the ensuing possibilities for introducing temporal control on the activity.Considering that the precatalyst Ru2 can be made from RuII arene sandwich complexes of type [Cp*Ru(arene)]X by UV irradiation in acetonitrile,[15,24] we anticipated that the catalytic species resulting from mixing Ru2 with water could be equally generated from these RuII sandwich complexes,provided that the arene ligand could be easily released with light under aqueous conditions.This idea was attractive not only as ameans to control reactivity,but also because of the high stability of the ruthenium(II) sandwich precursors,which might be especially useful to avoid its deactivation in biological contexts. We therefore synthetized the naphthalene derivative [Cp*Ru(naphthalene)]BPh4(Ru3), and two analogs with pyrene ligands:[Cp*Ru(pyrene)]PF6(Ru4)and [Cp*Ru(pyrene-SO3Na)]PF6(Ru5).[24] Their potential as photoactivatable catalysts for the RuAtACwas tested in amodel reaction using thioalkyne 2a and the p-tolyl azide 1b (Table 3).[25] Not surprisingly,treatment of 1b and 2awith 5mol%ofRu3,in a9:1 water:CH3CN mixture,did only provide traces of the triazole product (<3% yield, Table 3, entry 1).[23] However, we were glad to observe that when this mixture was irradiated with a365 nm LED lamp for 10 min, the cycloaddition Scheme 1. Scope of the annulation using the complex Ru2.[a] The counterion is probably bromide, as in the starting material. [b] Yield of the reaction carried out using aH 2O/DMSO (9:1) mixture is shown in parenthesis. [c] Carried out using Ru1 instead of Ru2.Fmoc=fluorenylmethoxycarbonyl. Table 2: Orthogonality between RuAtAC and CuAAC annulations.[a] Entry Catalyst (reaction time) Yield [%][b] 3ba3bh 1[Cu] (2 h) 078 2[c] [Cu] (2 h);then [Ru] (2 h)[c] 79 78 3[Ru] (2 h) 79 0 4[c] [Ru] (2 h);then [Cu] (2 h)[c] 78 95 5[d] [Ru] and [Cu] (2 h) 44 50 [a] Reaction conditions: Asolution of 2a (2.0 equiv), 2h (2.0 equiv), 1b (1.0 equiv;0.75 mmolg@1)inDMSO was added to water (500 mL, 75 mM), followed by the corresponding catalyst:either [Ru] (corresponds to Ru2, 5mol%) or [Cu] (correspondstoCuSO4·5H2O, 5mol%, NaAsc, 10 mol%), in avial open to air.[b] Yield determined by 1HNMR spectroscopy using 1,3,5-trimethoxybenzene as an internal standard. [c] After 2h,asecond equivalent of 1b was added,followed by the second catalyst. [d] Both [Ru] and [Cu] were present from the beginning. A ngewandte Chemie Research Articles 16061Angew.Chem. Int.Ed. 2021,60,16059–16066 T2021 TheAuthors.AngewandteChemieInternational Editionpublished by Wiley-VCHGmbH www.angewandte.org proceeded smoothly to afford the desired triazole 3bain 90% yield (entry 2). Therelated pyrene complex Ru4 was slightly more efficient, providing a99% yield upon photoactivation (10 min/ 365 nm, entry 4). In absence of light there is no conversion (entry 3). Thesulfonate derivative Ru5 also works under irradiation, although it is less efficient. With these results in hand, we next studied the behavior of precatalysts Ru2, Ru4 and Ru5 under more diluted conditions.Tocarry out these assays we used as substrate the water soluble triphenylphosphonium-containing azide 1c,because the phosphonium tag facilitates ahighly precise monitoring by LC–ESI-MS.For comparative purposes,wealso analyzed the performance of our first generation catalyst [Cp*RuCl- (COD)] (Ru1), which had been poorly effective at the micromolar range.Reactions were carried out in water, at rt for 4h,using different concentrations of the azide 1c(1 mM, 500 mM, 250 mMand 100 mM) and 50 mol%ofthe ruthenium complexes (Figure 3and Figure S5). In the case of the photoactivatable complexes Ru4 and Ru5,the reaction mixture in water, without any cosolvent,[26] was irradiated for 15 min with a365 nm LED.Gratifyingly,ascan be seen in the Figure 3, the three ruthenium complexes provided quantitative yields of the product when using azide concentrations of 1mMand 500 mM(after 4h), whereas the chloride ruthenium complex Ru1 provided only amodest 35%yield with an azide concentration of 1mM, or very poor yields at higher dilutions (2–7%yield). Theresults at 250 mMshowed that the pyrene RuII complex Ru4 is the most active, producing aquantitative yield of 3ca,while the analogue complex bearing asulfonate moiety (Ru5)led to amoderate 67%yield. Thetris(acetonitrile)ruthenium(II) catalyst (Ru2) and the photoactivatable pyrene complex Ru4 were the most effective catalysts when the concentration of the azide was decreased down to 100 mM(30 and 47%yield, respectively). Theamount of Ru2 and Ru4 can be decreased up 15 mol% without significantly eroding the efficiency(52 and 84%yield at 250 mM, respectively with Ru2 and Ru4;see Figures S6 and S7).[27] As indicated in Scheme 2, the reaction under these diluted conditions is not limited to 1c and 2a;other azides and thioalkynes are also suitable reactants,confirming the potential of the methodology,either using the precatalyst Ru2 or the light-activatable precursor Ru4 (Scheme 2). At this stage,wemoved to more demanding,biologically relevant environments,using a500 mMconcentration of the azide and 50 mol%ofthe ruthenium complexes (Figure 4). Gratifyingly,the cationic complex Ru2 provided very good results regardless of the biological media used. Thus,yields above 94%were obtained in PBS,cell culture milieu (DMEM), both with and without Fetal Bovine Serum, and even in presence of HeLa cell lysates.The photoactivatable complex Ru4 also showed an excellent performance under Figure 3. Catalyst performance in the micromolar range. Reactions were conducted in HPLC vials, and yields were determined by UHPLC– MS using coumarin as an internal standard. Resultsare the average of three different reactions. Reaction mixtures in the presence of Ru4 and Ru5 were irradiated for 15 min at 365 nm to activate the catalyst. Controls without irradiation for Ru4 and Ru5 provided yields <1%. Note:The counterion in 1c is Br@. Scheme 2. Scope of the reaction at micromolar concentrations (1: 250 mM; 2:500 mM). Reactionswere conducted in HPLC vials open to air and yields were determined by UHPLC–MS, using coumarin as an internal standard. Resultsare the average of three different reactions. Reactionsusing Ru4 were irradiated for 15 min at 365 nm. Table 3: Viability of the cycloaddition with phototoactivated Cp*Ru arene complexes Ru3,Ru4 and Ru5.[a] Entry [Ru] Solvent Conv.[%][b] Yield [%][b] 1Ru3 H2O/MeCN (9:1) 33 2Ru3+hnH2O/MeCN (9:1) 90 90 3Ru4[c] H2O/MeCN (9:1) 10 4Ru4+hnH2O/MeCN (9:1) 99 99 5Ru5+hnH2O/MeCN (9:1) 80 70 [a] Conditions for the reaction under irradiation: 2a (2.0 equiv), 1b (1.0 equiv), the solvent and [Ru] were sequentially added to avial, which was closed and irradiated with a365 nm LED lamp for 10 min, and the mixture stirred for 2h.[b] Yield and conversion determined by 1HNMR spectroscopy using 1,3,5-trimethoxybenzene as an internal standard. [c] The reaction was carried out without irradiation. A ngewandte Chemie Research Articles 16062 www.angewandte.org T2021 TheAuthors.AngewandteChemieInternational Edition publishedbyWiley-VCH GmbH Angew.Chem. Int.Ed. 2021,60,16059–16066 light irradiation, providing quantitative yields in PBS as well as in Hela cell lysates,and good yields,from 59 to 70%, in cell culture media. Curiously, Ru5 (with light) performed worse than Ru4 and Ru2. To better assess the potential of these catalytic systems,we compared their performance with that of Rhodium and Iridium complexes previously developed for related azide– alkyne annulations.[13,14] Therefore,wetested the reaction of 2aand 1c in water (at 250 mM), as well as in the presence of cell culture media (at 500 mM), in both cases using 50 mol% of the metal complexes.Reactions with Rh and Ir complexes were carried out using the azide:thioalkyne ratio that had been identified as optimal for each of these metal catalysts (azide:thioalkyne =1.5:1 for Ir and 1:2for Rh).[23] As can be deduced from Figure 5, [Ir(COD)Cl]2afforded the desired product in water in 46%yield, whereas [Rh(CO2)Cl]2only provided a24% yield of 3ca. Unfortunately,the performances of these metals dropped dramatically when used in biologically complex media such as DMEM or HeLa Cell lysates (Figure 5and Figure S10). Therefore,our ruthenium complexes are clearly superior in water and, especially under biologically demanding conditions. Aparticularly appealing application of bioorthogonal chemistry is related with the chemoselective modification of biopolymers.Therefore,weanalyzed whether our catalysts could also be used for bioconjugation reactions with peptides or nucleic acids.Asindicated in Scheme 3, the trisacetonitrile RuII complex Ru2 is effective in promoting the cycloaddition between the thioalkyne 2a and an heptapeptide bearing a6- azidolysine at the N-terminal position, to yield the corresponding peptide–triazole in 55%yield [Scheme 3, Eq. (1)]. More importantly,when this reaction was carried out with the photoactivatable catalyst Ru4,under 15 min irradiation at 365 nm, the reaction proceeded even more efficiently,providing the desired peptide conjugate in an excellent 84%yield. In the case of assDNA, we found that oligonucleotides containing an azide-modified adenine at its 5’end [Scheme 3, Eq. (2)],can be readily labelled with athioalkyne derivative Figure 4. Reactivity of Ru2,Ru4 and Ru5 under biologically relevant conditionsat500 mM(1c). Reactionswere conducted in HPLC vials. Reaction yields were measured by UHPLC–MS using an internal standard (coumarin). Reactionswith Ru4 and Ru5 were carried out by irradiatingthe mixturesfor 15 min at 365 nm to activate the catalyst. PBS=phosphate buffer solution;DMEM=Dulbecco’s Modified Eagle Medium;DMEM*=DMEM+10%fetal bovine serum+1% antibiotics;DEMEM-HEPES=DMEM without phenol red and with HEPES (4- (2-hydroxyethyl)-1-piperazineethanesulfonic acid);Lysates=HeLa cell lysate 5mgmL@1. Figure 5. Comparison of different metal catalysts in the model reaction. In all examples, 50 mol%ofthe metal complex was used. Ir stands for [Ir(COD)Cl]2;Rh stands for [Rh(CO2)Cl]2.Reactions were performed using the optimal azide/thioalkyne ratios found for each metal catalyst (1.5:1 for Ir and 1:2for Rh and Ru complexes). Yields were determined by UHPLC–MS using coumarin as an internal standard. Scheme 3. DNA and peptide labeling using Ru2 and Ru4 catalysts. A ngewandte Chemie Research Articles 16063Angew.Chem. Int.Ed. 2021,60,16059–16066 T2021 TheAuthors.AngewandteChemieInternational Editionpublished by Wiley-VCHGmbH www.angewandte.org equipped with aRhodamine tag, using either Ru2 and Ru4 (with irradiation), to give the expected products in 50 and 72%yields,respectively.Therefore,again, the photoactivatable catalyst Ru4 is the one presenting the best performance. As afinal test of the potential of our new catalytic systems, we checked the viability of the annulations in cellular environments,using the phosphonium azide 1c.The reaction could be monitored by LC–MS,owing to the sensitivity enhancement offered by phosphonium cations in mass spectrometry (Figure 6).[23] Theexperiments were carried out by mixing the ruthenium catalyst (Ru2 or Ru4), the thioalkyne (2a,800 mM) and azide 1c (100 mM) in DMEM– HEPES containing HeLa cells (1 X106cellsmL@1). After 2h, the cells were centrifuged, the supernatant collected, and the cell pellets treated with MeOH (80%aq.) to extract the cellular content. Both the extracellular media and the methanol extracts were analyzed by LC–MS.Either using Ru2,orthe photoactivatable complex Ru4 (with irradiation for 15 min), we were glad to detect the expected reaction product 3ca,both in the supernatant as well as in the methanolic extract.[28] Worth to note,the methanolic extract turned out to be particularly rich in triazole product 3ca, whereas the supernatant contained considerable amounts of both azide (1c)and triazole (3ca). However,itislikely that at least part of the product internalizes after being formed. Overall, both, the photoactivatable complex Ru4 and the cationic Ru2,were capable to promote the annulation under diluted conditions in cellular suspensions. Conclusion We have discovered that specifically tailored cationic ruthenium (II) complexes are highly effective precatalysts to perform Ruthenium Catalyzed Azide Thioalkyne Cycloadditions (RuAtAC) in aqueous media. These precatalysts allow to carry out the annulation under dilute conditions,and also in complex, biorelevant media. Moreover,the reaction is fully compatible and mutually orthogonal to the standard CuAAC. Importantly,the ruthenium reagents can be engineered as arene sandwich complexes to work as light-activatable precatalysts.This provides not only for the temporal control of the reactivity,but also for higher efficiencies,likely because of the intrinsic stability of the metal complexes until receiving the optical stimulus.We have also demonstrated that the ruthenium complexes can be used for the orthogonal modification of azide-tagged peptides and oligonucleotides,and that the reaction can be carried out in the presence of cells. Acknowledgements This research received financial support from the Spanish MINECO (SAF2016-76689-R, CTQ2017-84767-P and PID2019-106184GB-I00, and aFPU predoctoral Fellowship to AGG), the Xunta de Galicia (ED431C 2017/19, 2015- CP082, Centro Singular de InvestigacilndeGalicia accreditation 2019–2022, ED431G 2019/03), the ERDF,and the ERC (Adv.Grant No.340055). TheOrfeo-Cinqa network (CTQ2016-81797-REDC) is also acknowledged. We thank J. Miguel Avila and R. Menaya for their assistance with experiments in the presence of Hela cells. Conflict of interest Theauthors declare no conflict of interest. Keywords: biocompatible reactions ·bioorthogonal reactions · click chemistry ·ruthenium ·thioalkynes [1] Forreviews on bioorthogonal transformations,see:a)E.M. Sletten, C. R. Bertozzi, Angew.Chem. Int. Ed. 2009,48,6974– 6998; Angew.Chem. 2009,121,7108 –7133;b)H.W.Shih, D. N. Kamber,J.A.Prescher, Curr.Opin. Chem. Biol. 2014,21,103– 111;c)E.Brachet, P. Belmont, Curr.Org.Chem. 2016,20,2136– 2160;d)X.Ji, Z. Pan, B. Yu,L.K.DelaCruz, Y. Zheng,B.Ke, B. Wang, Chem. Soc.Rev. 2019,48,1077 –1094;e)H.Wu, N. K. Devaraj in Cycloadditions in Bioorthogonal Chemistry (Eds.:M. Vrabel, T. Carell), Springer International Publishing,Cham, 2016,pp. 109 –130;f)D.M.Patterson, J. A. Prescher, Curr. Opin. Chem. Biol. 2015,28,141 –149;g)J.M.Baskin, C. R. Bertozzi, Aldrichimica Acta 2010,43,15–23;h)D.M.Patterson, L. A. Nazarova, J. A. Prescher, ACSChem. Biol. 2014,9,592 – 605;i)R.D.Row,J.A.Prescher, Acc.Chem. Res. 2018,51, 1073–1081;j)N.K.Devaraj, ACSCent. Sci. 2018,4,952–959; k) M. Tom#s-Gamasa, J. L. MascareÇas, ChemBioChem 2020, 21,294–309. Figure 6. Reaction in the presence of HeLa cells suspended in DMEMHEPES. HPLC traces:MSsignal of the methanolic cell extract (CE) and supernatant (S) for a) Ru2,b)Ru4 (with 15 min irradiation), and c) Ru4 without irradiation.See the SupportingInformation for further details and control experiments. A ngewandte Chemie Research Articles 16064 www.angewandte.org T2021 TheAuthors.AngewandteChemieInternational Edition publishedbyWiley-VCH GmbH Angew.Chem. Int.Ed. 2021,60,16059–16066 [2] Forselected applications, see:a)M.Grammel, H. C. Hang, Nat. Chem. Biol. 2013,9,475–484;b)P.Agarwal, C. R. Bertozzi, Bioconjugate Chem. 2015,26,176–192. [3] Forreviews covering recent advances,see:a)J.J.Soldevila- Barreda, N. Metzler-Nolte, Chem. Rev. 2019,119,829–869; b) K. Vong,K.Tanaka in Handbook of In Vivo Chemistryin Mice:FromLab to Living System,Wiley-VCH, Weinheim, 2020, pp.309–353;c)P.Destito,C.Vidal, F. Llpez, J. L. MascareÇas, Chem. Eur.J.2021,27,4789–4816;d)P.K.Sasmal, C. N. Streu, E. Meggers, Chem. Commun. 2013,49,1581–1587;e)M.Yang, Y. Yang,P.R.Chen, Top. Curr.Chem. 2016,374,2;f)E.V. Vinogradova, Pure Appl. Chem. 2017,89,1619–1640;g)M. Mart&nez-Calvo,J.L.MascareÇas, Coord.Chem. Rev. 2018,359, 57–79; h) A. H. Ngo,S.Bose,L.H.Do, Chem. Eur.J.2018,24, 10584–10594;i)M.Mart&nez-Calvo,J.L.MascareÇas, Chimia 2018,72,791–801. [4] Forselected recent examples,see:a)D.Chang,E.Lindberg,S. Feng,S.Angerani, H. Riezman, N. Winssinger, Angew.Chem. Int. Ed. 2019,58,16033–16037; Angew.Chem. 2019,131,16179– 16183;b)P.Destito,A.Sousa-Castillo,J.R.Couceiro,F.Llpez, M. A. Correa-Duarte,J.L.MascareÇas, Chem. Sci. 2019,10, 2598–2603;c)C.Vidal, M. Tom#s-Gamasa, A. Guti8rrez- Gonz#lez, J. L. MascareÇas, J. Am. Chem. Soc. 2019,141, 5125–5129;d)X.Wang,Y.Liu, X. Fan, J. Wang,W.S.C.Ngai, H. Zhang,J.Li, G. Zhang,J.Lin, P. R. Chen, J. Am. Chem. Soc. 2019,141,17133–17141;e)R.Das,R.F.Landis,G.Y.Tonga, R. Cao-Mil#n, D. C. Luther,V.M.Rotello, ACSNano 2019,13, 229–235;f)S.Learte-Aymam&,C.Vidal, A. Guti8rrez-Gon- z#lez, J. L. MascareÇas, Angew.Chem. Int. Ed. 2020,59,9149– 9154; Angew.Chem. 2020,132,9234–9239;g)J.Miguel-_vila, M. Tom#s-Gamasa, J. L. MascareÇas, Angew.Chem. Int. Ed. 2020,59,17628–17633; Angew.Chem. 2020,132,17781–17786; h) B. L. Oliveira, B. J. Stenton, V. B. Unnikrishnan, C. R. de Almeida, J. Conde,M.Negr¼o, F. S. S. Schneider, C. Cordeiro, M. G. Ferreira, G. F. Caramori,J.B.Domingos, R. Fior, G. J. L. Bernardes, J. Am. Chem. Soc. 2020,142,10869–10880;i)A.M. P8rez-Llpez, B. Rubio-Ruiz, T. Valero,R.Contreras-Montoya, L. _lvarez de Cienfuegos,V.Sebasti#n, J. Santamar&a, A. Unciti- Broceta, J. Med. Chem. 2020,63,9650–9659;j)J.Ceballos,E. Grinhagena, G. Sangouard, C. Heinis,J.Waser, Angew.Chem. Int. Ed. 2021,60,9022–9031; Angew.Chem. 2021,133,9104– 9113;k)C.Vidal, M. Tom#s-Gamasa, P. Destito,F.Llpez, J. L. MascareÇas, Nat. Commun. 2018,9,1913;l)M.Mart&nez-Calvo, J. R. Couceiro,P.Destito,J.Rodr&guez, J. Mosquera,J.L. MascareÇas, ACSCatal. 2018,8,6055–6061. [5] a) H. C. Kolb,M.G.Finn, K. B. Sharpless, Angew.Chem. Int. Ed. 2001,40,2004–2021; Angew.Chem. 2001,113,2056 –2075; b) C. W. Tornøe,C.Christensen,M.Meldal, J. Org. Chem. 2002, 67,3057–3064. [6] a) J. E. Hein, V. V. Fokin, Chem. Soc.Rev. 2010,39,1302–1315; b) V. K. Tiwari, B. B. Mishra, K. B. Mishra, N. Mishra, A. S. Singh, X. Chen, Chem. Rev. 2016,116,3086–3240;c)D.Schulz, A. Rentmeister, ChemBioChem 2014,15,2342–2347;d)F. Musumeci, S. Schenone,A.Desogus,E.Nieddu, D. Deodato,L. Botta, Curr.Med. Chem. 2015,22,2022–2050;e)J.Matyas ˇovsky ´,P.Perlikova, V. Malnuit, R. Pohl, M. Hocek, Angew.Chem. Int. Ed. 2016,55,15856–15859; Angew.Chem. 2016,128,16088– 16091. [7] V. Hong, N. F. Steinmetz, M. Manchester, M. G. Finn, Bioconjugate Chem. 2010,21,1912–1916. [8] D. C. Kennedy,C.S.McKay,M.C.B.Legault, D. C. Danielson, J. A. Blake,A.F.Pegoraro,A.Stolow,Z.Mester,J.P.Pezacki, J. Am. Chem. Soc. 2011,133,17993. [9] Forthe use of stabilizing ligands to improve efficiencyand reduce toxicity,see:a)V.O.Rodionov, S. I. Presolski, D. D. Diaz, V. V. Fokin, M. G. Finn, J. Am. Chem. Soc. 2007,129, 12705–12712;b)C.Besanceney-Webler,H.Jiang,T.Zheng,L. Feng,D.Soriano del Amo,W.Wang,L.M.Klivansky,F.L. Marlow,Y.Liu, P. Wu, Angew.Chem. Int. Ed. 2011,50,8051– 8056; Angew.Chem. 2011,123,8201–8206;c)C.Uttamapinant, A. Tangpeerachaikul, S. Grecian, S. Clarke,U.Singh, P. Slade, K. R. Gee,A.Y.Ting, Angew.Chem. Int. Ed. 2012,51,5852– 5856; Angew.Chem. 2012,124,5954–5958;copper-free,strainpromotedannulations have been shown to be an efficient alternative;however,these reactions also present limitations associatedwith the side reactivity of reactants;see:d)J.C. Jewett, C. R. Bertozzi, Chem. Soc.Rev. 2010,39,1272–1279. [10] Forrecent examples of alternative Cu catalystsfor CuAAC, see: a) Y. Bai, X. Feng,H.Xing,Y.Xu, B. K. Kim, N. Baig,T.Zhou, A. A. Gewirth, Y. Lu, E. Oldfield, S. C. Zimmerman, J. Am. Chem. Soc. 2016,138,11077–11080;b)J.Chen, J. Wang,Y.Bai, K. Li, E. S. Garcia, A. L. Ferguson, S. C. Zimmerman, J. Am. Chem. Soc. 2018,140,13695–13702;c)J.Chen, J. Wang,K.Li, Y. Wang,M.Gruebele,A.L.Ferguson, S. C. Zimmerman, J. Am. Chem. Soc. 2019,141,9693–9700;d)J.Chen, K. Li, J. S. L. Shon, S. C. Zimmerman, J. Am. Chem. Soc. 2020,142,4565–4569; e) Q. Lu, S. Bai, Z. Chen, N. Zheng,X.Feng,Y.Bai, ACSMater. Lett. 2020,2,89–94;f)J.Clavadetscher, S. Hoffmann, A. Lilienkampf,L.Mackay,R.M.Yusop,S.A.Rider,J.J.Mullins, M. Bradley, Angew.Chem. Int. Ed. 2016,55,15662–15666; Angew.Chem. 2016,128,15891–15895;g)F.Wang,Y.Zhang,Z. Liu, Z. Du, L. Zhang,J.Ren, X. Qu, Angew.Chem. Int. Ed. 2019, 58,6987–6992; Angew.Chem. 2019,131,7061 –7066;h)Y.You, F. Cao,Y.Zhao,Q.Deng,Y.Sang,Y.Li, K. Dong,J.Ren, X. Qu, ACSNano 2020,14,4178–4187;i)J.Huang,L.Wang,P.Zhao,F. Xiang,J.Liu, S. Zhang, ACSCatal. 2018,8,5941–5946;j)S.Li, L. Wang,F.Yu, Z. Zhu, D. Shobaki, H. Chen, M. Wang,J.Wang, G. Qin, U. J. Erasquin, L. Ren, Y. Wang,C.Cai, Chem. Sci. 2017, 8,2107 –2114;k)L.Xue,Y.Yang,S.Wu, Y. Huang,J.Li, Y. Xiang,G.Li, Anal. Chem. 2020,92,2972–2978;l)J.Miguel- _vila, M. Tom#s-Gamasa, A. Olmos,P.Perez, J. L. MascareÇas, Chem. Sci. 2018,9,1947–1952. [11] P. Destito,J.R.Couceiro,H.Faustino,F.Llpez, J. L. MascareÇas, Angew.Chem. Int. Ed. 2017,56,10766–10770; Angew. Chem. 2017,129,10906–10910. [12] W. G. Kim, M. E. Kang,J.B.Lee,M.H.Jeon, S. Lee,J.Lee,B. Choi, P. M. S. D. Cal, S. Kang,J.-M. Kee,G.J.L.Bernardes,J.-U. Rohde,W.Choe,S.Y.Hong, J. Am. Chem. Soc. 2017,139, 12121–12124. [13] a) Y. Liao,Q.Lu, G. Chen, Y. Yu,C.Li, X. Huang, ACSCatal. 2017,7,7529–7534;b)W.Song,N.Zheng,M.Li, K. Dong, J. Li, K. Ullah, Y. Zheng, Org.Lett. 2018,20,6705–6709;c)W.Song, N. Zheng,M.Li, K. Ullah, Y. Zheng, Adv.Synth. Catal. 2018, 360,2429–2434. [14] a) W. Song,N.Zheng, Org.Lett. 2017,19,6200 –6203;b)R. Chen, L. Zeng,Z.Lai, S. Cui, Adv.Synth. Catal. 2019,361,989– 994;c)L.Zeng,Z.Lai, C. Zhang,H.Xie,S.Cui, Org. Lett. 2020, 22,2220 –2224;d)M.Li, N. Zheng,J.Li, Y. Zheng,W.Song, Green Chem. 2020,22,2394–2398. [15] A. M. McNair,K.R.Mann, Inorg.Chem. 1986,25,2519 –2527. [16] Foraphotoinitiated CuAACwith temporal control, which so far has not been shown to be biocompatible,see:a)B.J.Adzima, Y. Tao, C. J. Kloxin, C. A. DeForest, K. S. Anseth, C. N. Bowman, Nat. Chem. 2011,3,256–259;See also:b)P.K.Sasmal, S. Carregal-Romero,W.J.Parak, E. Meggers, Organometallics 2012,31,5968–5970. [17] Previous studies had indicated that cationic Cp*RuII complexes such as Ru2 were not suitablecatalysts for promotingazide– (thio)alkyne cycloadditionreactions in organic solvents:a)B.C. Boren, S. Narayan, L. K. Rasmussen, L. Zhang,H.Zhao,Z.Lin, G. Jia, V. V. Fokin, J. Am. Chem. Soc. 2008,130,8923–8930; b) S. Ding,G.Jia, J. Sun, Angew.Chem. Int. Ed. 2014,53,1877 – 1880; Angew.Chem. 2014,126,1908–1911. A ngewandte Chemie Research Articles 16065Angew.Chem. Int.Ed. 2021,60,16059–16066 T2021 TheAuthors.AngewandteChemieInternational Editionpublished by Wiley-VCHGmbH www.angewandte.org [18] a) Complex Ru2’’can be independently prepared in quantitative yield by mixing Ru2 and the thioalkyne 2a in CH2Cl2at room temperature.See the SupportingInformationfor its preparation, X-ray crystallographic analysis and apreliminarymechanistic proposal. b) This type of (2+2+2) thioalkyne trimerization, promotedbyRu2,was not observed when the neutral RuII chloride complex Ru1 was used. [19] Forthe use of achloride source in combinationwith Ru2 in organic solvents, see:a)J.A.Varela, S. G. Rub&n, C. Gonz#lez- Rodr&guez, L. Castedo,C.Sa#,J. Am. Chem. Soc. 2006,128, 9262–9263;b)J.A.Varela, L. Castedo,C.Sa#,Org.Lett. 2003, 5,2841–2844. [20] Forprecedent on CpRu oxo complexes,see:a)R.Shimogawa, T. Takao,H.Suzuki, Organometallics 2014,33,289–301;b)S. Takemoto,H.Ishii, M. Yamaguchi, A. Teramoto, M. Tsujita, D. Ozeki, H. Matsuzaka, Organometallics 2019,38,4298 –4306; c) K. M. Rao,C.L.Day,R.A. Jacobson, R. J. Angelici, Organometallics 1992,11,2303–2304;d)L.Fan, M. L. Turner,M.B. Hursthouse,K.M.A.Malik, O. V. Gusev,P.M.Maitlis, J. Am. Chem. Soc. 1994,116,385 –386;e)J.A.Gilbert, D. S. Eggleston, W. R. Murphy,Jr.,D.A.Geselowitz,S.W.Gersten, D. J. Hodgson, T. J. Meyer, J. Am. Chem. Soc. 1985,107,3855–3864;for reviews of organic chemistry in/on water,see:f)C.-J.Li, L. Chen, Chem. Soc.Rev. 2006,35,68–82;g)V.Chanda, V. Fokin, Chem. Rev. 2009,109,725–748. [21] Analysis by ESI-MS of the mixtures resulting from dissolving Ru2 in water allowed the detection of molecularions corresponding to oxo derivates,[Cp*Ru(O)]+,[Cp*Ru(O)(MeCN)2] and [Cp*Ru(O)2(MeCN)2],as well as hydroxo species [{Cp*Ru- (O)}2(OH)]+(see the Supporting Information,Figure S11). [22] L. Xu, S. L. Kuan, T. Weil, Angew.Chem. Int. Ed. 2021,https:// doi.org/10.1002/anie.202012034; Angew.Chem. 2021,https://doi. org/10.1002/ange.202012034. [23] See the SupportingInformationfor further details. [24] According to previous studies,the displacement is initiated by an h6!h4arene shift with concomitant addition of an external ligand, and subsequent release of the arene unit, see:a)D.S. Perekalin, A. R. Kudinov, Coord. Chem. Rev. 2014,276,153– 173;b)D.S.Perekalin, N. V. Shvydkiy,Y.V.Nelyubina, A. R. Kudinov, MendeleevCommun. 2015,25,29–31;c)J.A.S. Howell, N. F. Ashford, D. T. Dixon, J. C. Kola, T. A. Albright, S. K. Kang, Organometallics 1991,10,1852–1864. [25] a) Theuse of anthracenyl azide 1b is also possible,but the irradiation must be performedimmediately before the addition of 1b,toavoid the decomposition of the anthracenyl moiety by the light. b) See Ref.[16b] for the use of Ru4 in aphototriggered N-allylcarbamatte cleavage. [26] Control experiments confirmed that, under these dilute conditions the presence of acetonitrile as acosolventisdetrimental for the activity of the photoactivatable catalysts Ru4 and Ru5, probably owing to competing coordination to the Ru center, which slows down the rate of the process. [27] In all cases,control experiments with photoactivatable catalysts Ru4 and Ru5,without irradiation, provided negligibleyields of triazole products. [28] Control experiments with Ru2 confirmed its low toxicity towards HeLa cells (see the Supporting Information). However,the use of Ru4 and light under the conditionsofthe in vitro experiments (365 nm, 15 min) caused severe damage to the cells.Thus,invivo applications will require specific optimization, the use of alternative two-photonirradiation settings or the development of visible-light-activatable Ru catalysts. Manuscript received:March 15, 2021 Acceptedmanuscript online: May 10, 2021 Version of record online: June 16, 2021 A ngewandte Chemie Research Articles 16066 www.angewandte.org T2021 TheAuthors.AngewandteChemieInternational Edition publishedbyWiley-VCH GmbH Angew.Chem. Int.Ed. 2021,60,16059–16066