Ruthenium-Catalyzed Azide–Thioalkyne Cycloadditions in Aqueous Media: A Mild, Orthogonal, and Biocompatible Chemical Ligation

German Edition:DOI:10.1002/ange.201705006 Cycloaddition International Edition:DOI:10.1002/anie.201705006 Ruthenium-Catalyzed Azide–Thioalkyne CycloadditionsinAqueous Media:AMild, Orthogonal, and Biocompatible Chemical Ligation Paolo Destito,Jos8R. Couceiro,H8lio Faustino,Fernando Llpez,* and Jos8L. MascareÇas* Abstract: The development of efficient metal-promoted bioorthogonal ligations remains as amajor scientific challenge. Demonstrated herein is that azides undergo efficient and regioselective room-temperature annulations with thioalkynes in aqueous milieu when treated with catalytic amounts of asuitable ruthenium complex. The reaction is compatible with different biomolecules,and can be carried out in complex aqueous mixtures such as phosphate buffered saline,cell lysates,fetal bovine serum, and even living bacteria (E. coli). Importantly,the reaction is mutually compatible with the classical CuAAC. The copper-catalyzed azide–alkyne cycloaddition (CuAAC), paradigm of “click” chemistry,[1] can be considered among the most relevant chemical transformations discovered in the last decades,with countless applications in many areas of science.[2] Thebiological relevance of this reaction stems from its robustness and compatibility with aqueous media, as well as from its good bioorthogonality.[3] However,the transformation still presents important limitations.Thus,in addition to being fairly incompatible with thiols,the reaction is essentially restricted to terminal alkynes,aconsequence of amechanism which requires the formation of copper acetylide intermediates (Scheme 1a). An important additional drawback has to do with the side reactivity and toxicity of copper ions in biological contexts.[4] Furthermore,toreach efficient conversions in typically diluted biological settings, 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.[5] These issues have been partially addressed by using copper-stabilizing ligands which enhance the biocompatibility and kinetic of the reactions.[6,7] Copper-free,strainpromoted annulations have been shown to be an efficient alternative,[8] however, these reactions also present limitations associated to the side-reactivity of the reactants.Therefore,the development of new bioorthogonal and biocompatible reactions which address some of the above limitations remains as amajor challenge.[9] In particular, the discovery of robust and aqueous-compatible metal-catalyzed annulations, as alternatives to the CuAAC, represents ahighly appealing goal.[10] Several azide–alkyne cycloadditions using metals other than copper have been described in recent years,[11] but only the ruthenium variant (RuAAC)[12] has shown ameaningful scope (Scheme 1b).[13] In contrast to the CuAAC, which encompasses dinuclear copper intermediates such as Iand II,[14] the ruthenium-promoted reaction involves intermediate species like III,which evolve into IV by oxidative cyclometalation, and eventually to the triazole products.[15] In keeping with this scenario,the RuAAC, essentially developed in organic solvents,tolerates disubstituted alkynes but can produce mixtures of regioisomers.Probably,the notion that it is not compatible with water and air atmospheres has precluded more biofocused investigations.[16,13] Recent data suggest that some ruthenium complexes can promote the process in water, but the reactions require thermal activation and present alimited scope.[17] Herein, we demonstrate that certain ruthenium(II) complexes can indeed catalyze the cycloaddition between azides and alkynes in water, and at room temperature.Importantly, Scheme 1. Key mechanistic features of CuAAC and RuAAC. [*] P. Destito, Dr.J.R.Couceiro, Dr.H.Faustino,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:fernando.lop[email protected] [email protected] Dr.F.Lkpez Instituto de Qu&mica Org#nica General CSIC Juan de la Cierva 3, 28006 Madrid (Spain) Supportinginformation and the ORCID identification number(s) for the author(s) of this article can be found under: https://doi.org/10.1002/anie.201705006. T2017 The Authors. Published by Wiley-VCH Verlag GmbH &Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permitsuse and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. A ngewandte Chemie Communications 10766 T2017 The Authors. Published by Wiley-VCH VerlagGmbH &Co. KGaA,Weinheim Angew.Chem. Int. Ed. 2017,56,10766 –10770 the reaction is especially efficient when thioalkynes are used as reaction partners (Scheme 1c). Moreover,the process is mutually compatible with the CuAAC, tolerant to different types of biomolecules,including thiols,and can be carried out in either phosphate buffered saline,cell lysates,orcell culture media, even in presence of living bacteria (E. coli). At the outset, we were inspired by areport of Jia, Sun, and co-workers on an iridium-promoted azide–thioalkyne cycloaddition.[18] Although the method was developed in anhydrous CH2Cl2,anisolated example in water using benzyl azide caught our attention. Unfortunately,when we tested the reaction of the thioalkyne 2a with the fluorogenic anthracenyl-azide probe 1a,[19] theyield of the corresponding adducts (3aa/3aa’’)was modest (Table 1, entry 1).[20] Remarkably,w hen using Cp*Ru(cod)Cl as ac atalyst,[15a] we observed, after 24 hours,asubstantial formation of the desired cycloadducts with excellent regioselectivity (3aa/3aa’’=19:1, 58% combined yield, entry 2). This good result, together with the previously demonstrated biocompatibility of this type of ruthenium complex,[21] prompted us to further explore the process.Doubling the equivalents of 2a led to an excellent yield of 99%ofthe desired triazoles after 9hours of stirring at room temperature.Monitoring the reaction at different times confirmed the formation of the products in 78%yield after just 30 minutes,with the rate being then gradually reduced (entry 4).[22] Theperformance of [Ir(cod)Cl]2could not be improved by using 2equivalents of thioalkyne,(entry 5). And other ruthenium(II) catalysts,such as Cp*Ru(PPh3)2Cl,[12] RuH2(CO)(PPh3)3,[17b] were not efficient (entries 6and 7). In contrast, the tetramer [Cp*RuCl]4[23] was quite effective (entry 8). Thereaction between 1a and 2a,could also be carried out in CH2Cl2.However,obtaining good yields required the use of anhydrous solvent and inert atmospheres (entries 9vs. 10), which is not necessary in water. It looks like the aqueous solvent is somewhat precluding the ruthenium species from being rapidly deactivated.[24] Thecycloaddition is also feasible using atypical internal alkyne such as 2b,albeit somewhat slower (10%less conversion after 30 min), and it led to a5:1 mixture of regioisomers (Table 1, entry 11). Other alkynes such as 2c were unreactive under identical reaction conditions (entry 12). Thehigher reactivity of the thioalkyne partner was clearly visible in across-competition experiment:when the azide 1a was reacted with a1:1 mixture of 2a and 2b (2 equiv each), the triazole 3aa,arising from the cycloaddition with the thioalkyne was exclusively observed in 98% yield (entry 13). Interestingly,NMR analysis of the interaction between Cp*Ru(cod)Cl and the alkynes (in CD2Cl2) demonstrated that while 2a displaces the cod ligand at room temperature, 2bdoes not induce any change (see Pages S6–S9 of the Supporting Information). Analogous experiments using [Cp*RuCl]4and 2a,revealed rapid formation of anew complex identified as [Cp*Ru(2a)Cl],whereas with alkyne 2bno new ruthenium species could be detected, even after 3hours.[25] Thus,the good performance of thioalkynes might be in part related to their ability to strongly coordinate the Cp*RuCl moiety at room temperature.Additionally,the presence of the sulfur atom should also favor the formation of the required ruthenacyclic intermediate of type IV (Scheme 1b). With these reaction conditions in hand, we analyzed the scope of the method (RuAtAC). Despite the relatively poor water solubility of many of the azides and thioalkynes,the reactions proved to be general at room temperature,and the corresponding triazoles were obtained in good yields and with excellent regioselectivities (Table 2). Thus,aryl and aliphatic substituents either attached to the sulfur atom or to the terminal position of the alkyne were tolerated (e.g. 3aa–af). Terminal or trimethylsilyl-substituted thioalkynes (2g,2h) provided the corresponding adducts 3ag and 3ah in good yields.Importantly,not only the anthracenyl and benzyl azide (1a,1b)participated in the process,but aliphatic azides such as (2-azidoethyl)benzene (1c)or2-azidoethan-1-ol (1d)also reacted cleanly to provide the corresponding triazoles (3ca, 3ce,3de). p-Tolyl azide reacted with 2a to provide 3ea with amoderate 40%yield, avalue that could be improved up to 61%byusing [Cp*RuCl]4as acatalyst. Interestingly,different types of fluorophore-equippedtrisubstituted triazoles could be generated by either using adansyl-based azide (such as in 3fa)orbyincorporating acoumarin moiety,either as part of the thioalkyne (e.g. 3aj)orofthe organic azide (3ga and 3ha). Additionally,following work developed by Waser and co-workers,[26] 2-thioglucose and acysteine-containing dipeptide were selectively thio-alkynylated with EBX reagents. Gratifyingly,although the low water solubility of the thioTable 1: Identificationofreaction conditionsinwater.[a] Entry Cat. (Xmol%) 1a/2Solv. t [h] Conv [%][b] 3/3’’[b] Yield [%][b,c] 1[Ir(cod)Cl]2(2.5) 1:1 H2O24421:0 29 2Cp*Ru(cod)Cl (5) 1:1 H2O246519:1 58 3Cp*Ru(cod)Cl (5) 1:2 H2O999 19:1 99 4Cp*Ru(cod)Cl (5) 1:2 H2O0.5 80 19:1 78 5[Ir(cod)Cl]2(2.5) 1:2 H2O24361:0 20 6Cp*Ru(PPh3)2Cl(5) 1:2 H2O244723:1 17 7RuH2(CO)(PPh3)3(5) 1:2 H2O240 –0 8[Cp*RuCl]4(1.25) 1:2 H2O249914:1 99 9[d] Cp*Ru(cod)Cl (5) 1:2 CH2Cl229917:1 99 10 Cp*Ru(cod)Cl (5) 1:2 CH2Cl224418:1 37 11[e] Cp*Ru(cod)Cl (5) 1:2 H2O999 5:1 95[e] 12[f] Cp*Ru(cod)Cl (5) 1:2 H2O2410–<5[f] 13[g] Cp*Ru(cod)Cl (5) 1:4 H2O499 19:1[h] 98[h] [a] Unless otherwise noted, 2a (1–2 equiv),water,and 1a (1 equiv, 75 mm)were sequentially added under air to avial containing the catalyst (that had been kept under N2), and the mixture was stirred at RT. [b] Determined by 1HNMR spectroscopy of the crude reaction mixture with an internal standard. [c] Combined yield of 3/3’’.[d] Carried out under an inert atmosphere in anhydroussolvent. [e] Carried out with 2b. Products: 3ab/3ab’’.[f]Carried out with 2c.Products: 3ac/3ac’’.[g] Carried out using both 2aand 2b(2 equiv each). [h] Products: 3aaand 3aa’’. cod=1,5-cyclooctadiene. A ngewandte Chemie Communications 10767Angew.Chem. Int.Ed. 2017,56,10766 –10770 T2017 The Authors. Published by Wiley-VCH Verlag GmbH &Co. KGaA,Weinheim www.angewandte.org alkynylated protected dipeptide 2k demanded the use of small amounts of acosolvent (i.e CH2Cl2,5–10%vol.), its reaction with 1a and 1fproceeded efficiently,thus affording the desired products 3akand 3fkin good yields.The reaction between an alkynylated thioglucose and 1a took place smoothly in water to give the triazole 3al in 64%yield. We next explored the bioorthogonality of the chemistry by performing the reaction in either the presence of different biomolecular additives or under biologically relevant conditions (Table 3). Gratifyingly,the RuAtACcould be efficiently carried out in the presence of glutathione (20 fold excess respect to the [Ru];entry 1), different aminoacids (entry 2), and even in the presence of arandom miniprotein (entry 3). Thedesired triazole was obtained in moderate to excellent yields and similar regioselectivities compared to those obtained in pure water (>15:1). Thereaction could also be carried out in phosphate-buffered saline (PBS;entry 4). Additionally,frequently used culture cell media, such as DMEM, fetal bovine serum (FBS), and Hela cell lysates are also excellent reaction media, so the triazole 3aa was obtained in yields varying from 77 to 91%, (entries 5–7). Analogue reactions in these media between (2-azidoethyl)- benzene (1c)and the thioalkyne 2e gave good yields of 3ce (see Table S3). At this point, it was of interest to contrast the performances of the RuAtACand CuAACinwater to establish strengths and weaknesses of each method. Thus,wecarried out parallel experiments using (2-azidoethyl)benzene (1c; 75 mm), 5mol%ofeach catalyst, and either 2e (for Ru) or phenylacetylene (for Cu). While the ruthenium-promoted reaction was significantly faster than the copper counterpart, when using CuSO4and sodium ascorbate for the latter (60% vs.22% yield, after 2h), the CuAACbecame faster by including additives such as BTTAA (75%yield after 2h;see Table S4). Importantly,the CuAACfailed with internal alkynes,including thioalkynes like 2a and, not surprisingly, it is essentially inhibited in the presence of thiols like glutathione (0%yield after 24 h). In contrast, the RuAtAC works effectively even in the presence of a20-fold excess of glutathione (75%yield), and provides the products with both internal and terminal thioalkynes (as shown in Table 2). On the weak side,the efficiency of the RuAtACdecreases upon dilution (16%yield at 250 mm), while the ligand-accelerated CuAACprovided a57% yield under similar micromolar conditions (see Pages S11–S13). Thelack of reactivity of internal thioalkynes in the presence of copper catalysts suggested that the CuAACand the RuAtACcould be mutually orthogonal.[27] Gratifyingly, the engineered diyne 4was quantitatively converted into the bis(triazole) 5,without cross-reactivity,byperforming aCuAACwith the dansyl azide 1f,and subsequent in situ addition of the ruthenium catalyst and the anthracenyl azide 1a(1 equiv with respect to thioalkyne;Scheme 2). Considering the scarcity of mutually compatible bioorthogonal reacTable 2: Scope of the RuAtAC in water at room temperature.[a] [a] Reaction conditions: 2(2 equiv), water,and 1a(1 equiv,75mm)were sequentiallyadded under air to avial containing Cp*Ru(cod)Cl (5 mol%) which had been kept under N2.The vial was closed, and the mixture stirred at RT for 15–24 h. Regioselectivities (3/3’’)were >18:1 unless otherwise noted (determined by 1HNMR analysis of the crude reaction mixtures with an internal standard).Yield of isolated pure 3, unless otherwise noted. Yields of the reactions carried out in anhydrous CH2Cl2under an inert atmosphere are shown within brackets. [b] Yield of 3determined by 1HNMR analysis of the crude reaction mixture with an internal standard. [c] Corresponds to a18:1mixture of 3ca/3ca’’. [d] Carried out with [Cp*RuCl]4(1.25 mol%). Table 3: Analysis of the biocompatibility of the method.[a] Entry Conditions[b] Conv.[%][c] 3/3’[c] Yield [%][c,d] 1H 2O/glutathione 80 19:1 60 2H 2O/Hist +Fmoc-ala 93 18:1 82 3H 2O/Peptide 39 aa (0.5 mm)9923:1 98 4PBS 99 18:1 97 5cell cultured media (DMEM) 99 15:1 84 6fetal bovine serum (FBS) 88 17:1 77 7cell lysates (Hela) 99 16:1 91 [a] Reaction conditions: 2a (2 equiv) was added to asuspension of Cp*Ru(cod)Cl (5 mol%), 1a (1 equiv,75mm), and the additive, in the selected milieu, and the resulting mixture was stirred for 24 h. [b] The additives in entries 1–2 are in 20-fold excess with respect to the ruthenium catalyst. [c] Determined by 1HNMR analysis of the crude reaction mixture using an internal standard. [d] Combined yield of 3aa/ 3aa’’. A ngewandte Chemie Communications 10768 www.angewandte.org T2017 The Authors. Published by Wiley-VCH Verlag GmbH &Co. KGaA,Weinheim Angew.Chem. Int. Ed. 2017,56,10766 –10770 tions,the possibility of using both annulations in tandem in one pot is certainly promising.[27] Finally,and importantly,wefound that the RuAtACcan also be carried in the presence of bacteria (E. coli)without compromising their viability.Therefore,incubation of PBS containing Ecoli with 1a (1 mm), 2a (2 mm), and Cp*Ru- (cod)Cl (100 mm)led to arapid increase in the fluorescence. After 24 hours,centrifugation and analysis in aplate reader of both the extracellular supernatant and the methanol/water (8:2) extracts of the resulting bacteria pellet showed acombined increase in fluorescence of eight times with respect to controls (see Figure S29). Importantly,the fluorescence was mainly concentrated inside the bacteria, and the product 3aa was also detected by HPLC-ESI. Analysis of the optical density of the bacterial cultures revealed that neither the catalyst nor the reactants are meaningfully toxic (see Table S5). In summary,wehave discovered anew methodology to achieve catalytic,orthogonal chemical annulations in water, at room temperature.The reaction is promoted by specific ruthenium(II) catalysts,works efficiently with avariety of azides and thioalkynes,and can be carried out in presence of biomolecules (glutathione,aminoacids,peptides). Thereaction is also efficient in phosphate buffered saline,and in complex biological media such as cell lysates and fetal bovine serum, and even in presence of living bacteria. Importantly, the reaction is mutually compatible with the classical CuAAC, thus providing the option of tandem biorthogonal processes. Acknowledgments This work has received financial support from Spanish grants (SAF2016-76689-R and SAF2013-41943-R), the Xunta de Galicia (2015-CP082 and Centro Singular de Investigacilnde Galicia accreditation 2016-2019 ED431G/09), the European Union (European Regional Development Fund -ERDF), and the ERC (Adv.Grant 340055). We also thank the OrfeoCinqa network CTQ2016-81797-REDC,and T. Seedat and V. Fraga for preliminary experiments. Conflict of interest Theauthors declare no conflict of interest. Keywords: alkynes ·azides ·chemical ligations · click chemistry ·ruthenium Howtocite: Angew.Chem. Int. Ed. 2017,56,10766–10770 Angew.Chem. 2017,129,10906–10910 [1] H. C. Kolb,M.G.Finn, K. B. Sharpless, Angew.Chem. Int. Ed. 2001,40,2004; Angew.Chem. 2001,113,2056. [2] Forinstance,see:a)J.E.Hein, V. V. Fokin, Chem. Soc.Rev. 2010,39,1302;b)F.Musumeci, S. Schenone,A.Desogus,E. Nieddu, D. Deodato,L.Botta, Curr.Med. Chem. 2015,22,2022; c) T. Zhang,Z.Zheng,X.Cheng,X.Ding, Y. Peng, Prog. Chem. 2008,20,1090;d)J.Matyas ˇovsky ´,P.Perl&kov#,V.Malnuit, R. Pohl, M. Hocek, Angew.Chem. Int. Ed. 2016,55,15856; Angew. Chem. 2016,128,16088. [3] a) L. Li, Z. Zhang, Molecules 2016,21,1393;b)V.K.Tiwari, B. B. Mishra, K. B. Mishra, N. Mishra, A. S. Singh, X. Chen, Chem. Rev. 2016,116,3086;c)D.Schulz, A. 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Bonin, C. Chauvier, J. Waser, J. Am. Chem. Soc. 2014,136,16563. [27] D. M. Patterson, J. A. Prescher, Curr.Opin. Chem. Biol. 2015, 28,141. Manuscript received:May 15, 2017 Revised manuscript received: June 25, 2017 Acceptedmanuscript online: July 6, 2017 Version of record online: August 3, 2017 A ngewandte Chemie Communications 10770 www.angewandte.org T2017 The Authors. Published by Wiley-VCH Verlag GmbH &Co. KGaA,Weinheim Angew.Chem. Int. Ed. 2017,56,10766 –10770