Quaternization of Vinyl/Alkynyl Pyridine enables ultrafast cysteine‐selective protein modification and charge modulation

German Edition:DOI:10.1002/ange.201901405 Protein Modification International Edition:DOI:10.1002/anie.201901405 QuaternizationofVinyl/Alkynyl Pyridine Enables Ultrafast CysteineSelectiveProtein Modification and Charge Modulation Maria J. Matos+,Claudio D. Navo+,Tuuli Hakala+,Xhenti Ferhati+,Ana Guerreiro, David Hartmann, Barbara Bernardim, Kadi L. Saar,Ismael CompaÇln, Francisco Corzana,* Tuomas P. J. Knowles,* Gonzalo Jim8nez-Os8s,*and GonÅalo J. L. Bernardes* Abstract: Quaternized vinyland alkynyl-pyridine reagents were shown to react in an ultrafast and selective manner with several cysteine-tagged proteins at near-stoichiometric quantities.Wehave demonstrated that this method can effectively create ahomogenous antibody–drug conjugate that features aprecise drug-to-antibody ratio of 2, which was stable in human plasma and retained its specificity towards Her2+cells. Finally,the developed warhead introduces a +1charge to the overall net charge of the protein, whichenabled us to show that the electrophoretic mobility of the protein may be tuned through the simple attachment of aquaternized vinyl pyridinium reagent at the cysteine residues.Weanticipate the generalized use of quaternized vinyland alkynyl-pyridine reagents not only for bioconjugation, but also as warheads for covalent inhibition and as tools to profile cysteine reactivity. Chemical site-selective modification offers ameans to diversify the function and properties of the protein.[1] For example,byusing the targeting capabilities of an antibody,it is possible to covalently attach avery potent drug to the antibody through aprecise chemical reaction to shuttle this drug to aspecific tissue.[2] Thetoolbox of reactions for protein modification has expanded significantly in the last decade.[3] Of these,reactions that target proteinogenic amino acids seem particularly suitable to modify native proteins in the test tube.Lysine,[4] methionine,[5] tryptophan,[6] and the N-[7] and the C-terminus[8] may now be targeted by using avariety of approaches.However,cysteine[9] remains perhaps the residue of choice to produce functional and, in particular, clinically useful protein conjugates,namely antibody–drug conjugates (ADCs). This choice is aresult of the high nucleophilicity of the sulfhydryl side-chain combined with the low abundance of free cysteineresidues,since many cysteines are paired as structural disulfides.Thus,many research groups have focused on developing efficient methods to chemoselectively modify cysteine-tagged proteins.For example,electrophiles, such as carbonylacrylic acid reagents for Michael addition,[10] arylation reactions based on transition metals,[11] or aselective amino acid sequence,[12] or conjugate additions at dehydroalanine[13] formed from cysteine or thiol-yne reactions using cyclooctynes[14] have been developed. Each of these methods has relative advantages and disadvantages,but amethod based on the simple attachment of awarhead-like structure whose utility would go beyond bioconjugation and for example be used to tune protein pharmacokinetics,through changes in the overall net charge of aprotein, or be used to design cysteine covalent inhibitors is missing from the current toolbox. Herein, we report the computational chemistry assisted discovery of quaternization of the nitrogen of vinyland alkynyl pyridines to convert otherwise non-reactive reagents into ultrafast and chemoselective cysteine-modifying reagents.The utility of these reagents was demonstrated for bioconjugation and for modulation of electrophoretic mobility through charge incorporation by using microfluidics. Based on computational predictions,weidentified promising alkenes and alkynes through modelling the nucleophilic addition reaction between asimple thiolate or an amine with various electrophiles.Asaresult of the lower pKaof solventexposed thiols relative to amines,itis expected that at neutral or slightly basic pH alarger proportion of thiols would be deprotonated. Activation free energies (DG*)for the corresponding addition reactions were predicted by using quantum mechanical calculations (Figure 1a and b). 2-Vinyland 2ethynyl pyridines (compounds 1and 3)were calculated to be poorly reactive toward thiolates (DG*>24 kcalmol@1)and unreactive toward primary amines (DG*>31 kcalmol@1). [*] Dr.M.J.Matos,[+] T. Hakala,[+] D. Hartmann,Dr. B. Bernardim, K. L. Saar,Prof. T. P. J. Knowles, Dr.G.J.L.Bernardes Department of Chemistry,University of Cambridge Lensfield Road, CB2 1EW Cambridge (UK) E-mail:t[email protected] [email protected] A. Guerreiro,Dr. G. J. L. Bernardes Instituto de Medicina Molecular,Faculdade de Medicina, Universidade de Lisboa Avenida ProfessorEgas Moniz, 1649-028 Lisboa (Portugal) E-mail:g[email protected] Dr.C.D.Navo,[+] X. Ferhati,[+] I. CompaÇkn, Dr.F.Corzana, Dr.G.Jim8nez-Os8s Departamento de Qu&mica,Universidad de La Rioja, Centro de InvestigaciknenS&ntesis Qu&mica 26006 LogroÇo (Spain) E-mail:[email protected] Dr.C.D.Navo,[+] Dr.G.Jim8nez-Os8s CIC bioGUNE,Bizkaia Technology Park Building 801A, 48170 Derio (Spain) E-mail:g[email protected] [++]These authors contributed equally to this work. Supportinginformation and the ORCID identification number(s) for the author(s) of this article can be found under: https://doi.org/10.1002/anie.201901405. T2019 The Authors. Published by Wiley-VCH Verlag GmbH &Co. KGaA. This is an open access article under the terms of the Creative Commons AttributionLicense, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. A ngewandte Chemie Communications 6640 T2019 The Authors. Published by Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim Angew.Chem.Int. Ed. 2019,58,6640 –6644 However,when quaternized, the resulting N-methylpyridinium derivatives were calculated to be exceedingly reactive towards nucleophilic addition, with dramatically decreased activation free energies.For instance,2-vinyland 2-ethynylpyridiniums 2and 4were calculated to be more than abillion times more reactive with thiolates (DG*=10–12 kcalmol@1) than their non-quaternized analogues 1and 3,which shows predicted reactivities similar to those of maleimide[15] and carbonylacrylic derivatives[10] previously used for cysteine ligation. In all cases,acomplete selectivity for cysteine over lysine addition was predicted, as reflected by the nearly 8kcalmol@1higher activation barrier calculated for the latter. We validated our computational predictions by reacting stoichiometric amounts of minimal cysteine and lysine sidechain model compounds (that is 1-propanethiol and 1propylamine,respectively) with commercially available 1–3 and synthesized reagent 4(Figure 1c), and monitoring reaction with 1HNMR spectroscopy (3 mm,phosphate buffer in D2O, pH 7.6;see the Supporting Information for additional data and adetailed discussion on the effect of using aslightly more acidic pH). In agreement with theoretical predictions,the reactions with non-quaternized reagents 1and 3were very slow and gave 62 and 6% conversions, respectively,ofaddition products after 24 hours (Figure 1c, Entries 1and 3). On the contrary,pyridine N-methylated reagents 2and 4gave aconversion of 99%ofaddition product after 1hour with only 1equivalent of electrophile (Figure 1c,Entries 2and 4). 2-Alkynyl reagent 4gave a6:4 mixture of isomers as products,namely the cis and trans alkene adducts (See the Supporting Information for their structural characterization and thermal stability). Thekinetic profiles obtained with aprotected cysteine (N-acetylcysteine amide,Ac-Cys-NH2)were similar, albeit slightly faster than with 1-propanethiol (89%adduct formation in 24 hours with non-quaternized 1,and 93%adduct formation in <4min with quaternized 2;Figure 1c,Entries 5and 6). Also,in agreement with computational predictions,the fast kinetics observed with quaternized pyridinium reagents 2and 4were comparable to those of N-ethylmaleimide,[15] for which the reaction with 1-propanethiol and Ac-Cys-NH2were complete within the 4min necessary to obtain the first 1HNMR spectroscopic measurements,although the formation of an undetermined alkene by-product was observed in these reactions with maleimide (see the Supporting Information). With lysine side-chain mimic 1-propylamine (Figure 1c, Entry 7) and N-acetyllysine amide (Ac-Lys-NH2;Figure 1c, Entry 8) the reactions with quaternized 1-methyl-2-vinylpyridinium (2)were exceedingly slow (<6% adduct formation after 24 hours with 1equivalent of electrophile). This result agrees with the computationally predicted orthogonality of these electrophiles towards cysteines,with no expected reaction on typically solvent-exposed lysines.Itisinteresting to note that the terminal alkyne hydrogen in compounds 3 and 4quickly exchanged to deuterium in D2OatpH7.6, particularly in the case of quaternized 4,for which deuteration takes place within 4min. H/D exchange at the vinyl hydrogens of quaternized 2was also observed when [D7]DMF was used as aco-solvent. Accurate reaction rate constants could not be obtained for very reactive quaternized pyridiniums 2and 4,and Nethylmaleimide as aresult of the ultrafast kinetics observed under the reaction conditions used (3 mmwas the detection limit for 1HNMR spectroscopy in our experiments). Also,the high level of sensitivity to small reagent concentration changes on the observed rate for second-order reactions, further complicates the derivation of the rate constants under stoichiometric conditions.Furthermore,although alkynyl pyridinium 4was selective towards mono-functionalization under strict stoichiometric conditions,itwas found to undergo double thiol addition to the external position of the triple bond to give astable dithioacetal when aslight excess of the thiol was used (see the Supporting Information). Once the dramatic effect of pyridine quaternization in boosting the electrophilicity of reagents 1and 3was demonstrated and given the very similar and ultrafast reactivity of quaternized reagents 2and 4towards thiols,wedecided to use more convenient 1-methyl-2-vinylpyridinium 2for cysteine-selective modification on proteins (to avoid terminal alkyne deuteration and formation of mixtures of potentially reactive alkene isomers upon nucleophilic addition). Having demonstrated the superior reactivity of 2towards thiols on small molecule models,wedecided to test the use of vinyl pyridinium reagents towards cysteine-tagged proteins (Figure 2a). We selected four representative proteins,the C2A domain of Synaptotagmin-I, Annexin-V,ubiquitin, and albumin, which display either natural or engineered single surface-exposed cysteines (see the Supporting Information). In all cases,and with stoichiometric amounts or only asmall excess of 2(1 equiv for ubiquitin and albumin and 10 equiv for annexin-V and C2Am), under buffered (NaPipH 8.0, Figure 1. Computer-assisted evaluationand experimental validation of a) vinyland b) alkynyl pyridinium electrophile reagents 1–4for cysteine modification. c) Reaction of 1–4with stoichiometric amounts of various nucleophiles. A ngewandte Chemie Communications 6641Angew.Chem. Int.Ed. 2019,58,6640 –6644 T2019 The Authors. Published by Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim www.angewandte.org 50 mm)conditions for 1hour at 3788C, complete conversion into the corresponding thioether adduct was observed, as confirmed by LC-MS analysis,and in high yield (>95%), as shown by Bradford protein assay (Figure 2b-2e;see also the Supporting Information;noreaction was detected with either 1or 2under slightly acidic pH 5.5). Despite the use of 10 equiv to ensure efficient modification of even sterically crowded, and thus poorly deprotonated, cysteine residues, such as in the case of Annexin-V,nonon-specific reactions at lysine were detected. Other studies that use vinyl-substituted pyridine derivatives achieved amaximum of only 85% conversion after 24 hours,[16] which demonstrates well the effect of quaternization of the pyridine on the kinetics of the bioconjugation reaction. Furthermore,the final conjugates did not react with EllmanQsreagent [5,5’-dithiobis(2-nitrobenzoic acid)] that showed that all cysteine had been consumed (see the Supporting Information). Importantly, we verified that the formed thioether bond is stable towards potential thiol exchange reactions,and the conjugates were also fully stable in human plasma in buffered pH 5–8 solutions (see the Supporting Information). Next, we confirmed that the conditions used are mild and do not induce changes in both the structure or function of the native proteins.Inthe case of albumin, conjugate albumin-2showed no significant changes in its secondary structural content as determined by circular dichroism (CD;Figure 2f)and retained its ability to bind to the neonatal Fc receptor (FcRn) as determined by Surface Plasma Resonance (KD=11.8 mmfor albumin versus KD=8.88 mmfor albumin-2;Figure 2g). Together, our data demonstrates the generalization of amethod for cysteineselective protein modification based simply on the use of quaternized vinyl pyridinium reagents.The products are formed in high conversion and yield, are stable in human plasma, and both native structure and function activities are retained. Next, we decided to demonstrate that vinyl pyridinium reagents may be easily functionalized first through quaternization of the pyridine nitrogen with an alkyne linker followed by subsequent derivatization through CuI-catalyzed azide– alkyne cycloaddition reaction with asuitable synthetic motif that displays an azido group (see the Supporting Information). We chose to build aquaternized vinyl pyridinium reagent that features cytotoxic drug monomethyl auristatin E (MMAE;Figure 3a). By connecting the cysteine-selective reagent and the drug, we introduced the dipeptide valine– citrulline (ValCit), which is known to cleave on exposure to cathepsin B,[17] and the self-immolating spacer p-aminobenzyl carbamate (PABC).[18] This reagent 5was then conjugated to Thiomab,anHER2 targeting antibody that has been engineered to contain an additional cysteine residue at position 205 in each light-chain (Thiomab LC-V205C).[19] To our delight, the conjugation reaction proceeded to completion with 5equiv per light-chain of 5,within 1hour at 3788CinNaPi pH 8.0, 20 mmbuffer (Figure 3a). LC-MS analysis showed asingle modification in each-light chain and no modifications in the heavy-chain (Figure 3b)toform aconjugate with aprecise drug-to-antibody ratio of 2. Importantly,Thiomab-5 conjugate retained its specificity towards Her2 positive cells, as demonstrated by flow cytometry analysis (Figure 3cand d). Our data demonstrate the use of quaternized vinyl pyridinium Figure 2. Cysteine-selective protein modification.a)The reaction of acysteine-tagged protein with 2.General conditions: acysteine-tagged protein (10–20 mm)isreacted with 2(1–10 equiv) in NaPi(50 mm, pH 8.0) at 3788Cfor 1hour.b–e) ES-MS spectraofthe product of the reaction between b) C2Am, c) Annexin V, d) Ubiquitin, and e) albumin with 2(deconvoluted spectra;expected increase of 118 Da in the total protein mass). f)Comparative CD analysis of albumin and albumin-2. g) Surface plasma resonanceofbinding to the FcRn receptor. kDfor rHSA and rHSA-2. Figure 3. Cysteine-selective antibody modification.a)Schematic for the bioconjugation of 5to Thiomab and chemical structure of 5.b)ES-MS spectra of the lightand heavy-chain of Thiomab after conjugation with 5.c)Counter plot indicates the binding affinity of Thiomab-5to Her2 expressingSKBR3 cells at 50 nm.d)Percentage of SKBR3 cells bound to Thiomab-5at 50 nm.Trastuzumaband Thiomab were used as positive controls and IgG Isotype was used as negative control. A ngewandte Chemie Communications 6642 www.angewandte.org T2019 The Authors. Published by Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim Angew.Chem.Int. Ed. 2019,58,6640 –6644 reagents to efficiently generate homogenous and functional antibody conjugates. Theconjugation of aprotein can be monitored directly by following changes in its electrophoretic mobility because the quaternized vinyl pyridinium reagent introduces an additional charge of +1per cysteine residue to the net charge.For instance,albumin features one single free cysteine and thus conjugation of 2adds +1charge to the overall net charge of the protein (charge ca. @7atpH7). We decided to test this hypothesis by using amicrofluidic free-flow electrophoresis device,[20] which allows the charge of proteins to be determined under native conditions in solution and requires only low volumes of sample.The samples were introduced into the device with aflanking buffer, and the deflection of the protein samples was observed while atransverse electric field was applied by increasing the potential between electrodes in the device from 0–120 V(Figure 4a). When the measured deflection in the field was converted into drift velocity (Vdrift)and the electric field inside the devices was calibrated (Figure 4b), aclear difference in the electrophoretic behavior of albumin and albumin-2was observed. Furthermore,the electrophoretic mobilities were estimated to be: @1.71: 0.21X10@8m2V@1s@1to give acharge of about &@6.84: 0.58 and @1.12:0.10X10@8m2V@1s@1to give acharge of about @4.64:0.12 for albumin and albumin-2,respectively. Thedecrease in mobility can be explained by the direct proportionality of the charge (q)tothe electrophoretic mobility [mel;q=(mel XkBT)/D], which implies that if the charge is decreased, the mobility should also decrease as confirmed by the data. Moreover,because we are adding positive charge through conjugation of the quaternized vinyl pyridinium reagent 2into anegatively charged albumin, the net charge of conjugate albumin-2should be smaller, as observed in the results obtained with the microfluidic electrophoresis.These data thus demonstrate the application of site-selective protein modification to change the overall net charge of aprotein in acontrolled manner, and consequently modulate its electrophoretic mobility. In summary,anefficient and irreversible cysteine-selective bioconjugation method is reported. This method is enabled by the discovery that quaternization of the nitrogen of vinyland alkynyl pyridines transforms these molecules into extremely reactive electrophiles towards thiols.We demonstrate the utility of these reagents for cysteine-selective bioconjugation of five different protein scaffolds,including aclinically used antibody.Importantly,the conjugates formed are resistant towards thiol exchange reactions and retain their native activity.Byusing amicrofluidic setup,weshow that the electrophoretic mobility of aprotein may be modulated through simple attachment of the quaternized vinyl pyridinium reagent at cysteine to introduce a +1charge to the overall net charge of the protein. This property of the quaternized vinyl pyridinium may prove useful to enhance the hydrophilicity,half-life in circulation, or internalization rates of ADCs.The simplicity of these reagents combined with their excellent reactivity and selectivity towards thiols,makes them ideal for protein bioconjugation, but also for use as warheads in the design of cysteinecovalent ligands[22] or to probe hyper-reactive cysteines within the human proteome in chemical proteomic approaches.[23] Acknowledgements Funded under the EU Horizon 2020 Programme,Marie Skłodowska-Curie ITN GA No.675007, the Royal Society (UF110046 and URF\R\180019 to G.J.L.B.), FCT Portugal (iFCT IF/00624/2015 to G.J.L.B.and PhD studentship SFRH/ BD/115932/2016 to A.G.), Xunta de Galicia (Galician Plan of research, innovation and growth 2011–2015, ED481B 2014/ 086-0 and ED481B 2018/007 to M.J.M.), D.G.I. MINECO/ FEDER (grants CTQ2015-70524-R and RYC-2013–14706 to G.J.-O.and C.D.Nand CTQ2015-67727-R to F.C.), Universidad de la Rioja (FPI PhD studentship to I.C.), FAPESP (BEPE 2015/07509-1 and 2017/13168-8 to B.B.), and by an ERC StG (GA No.676832). We also thank Genentech for providing the Thiomab antibody,Albumedix for providing rHSA, Dr. Andr8Neves and Prof.Kevin Brindle for providing C2Am, and Dr. Vikki Cantrill for her help with the editing of this manuscript. Conflict of interest Theauthors declare no conflict of interest. Keywords: antibody–drug conjugates ·bioconjugation · cysteine ·microfluidics ·protein modification Howtocite: Angew.Chem. Int. Ed. 2019,58,6640–6644 Angew.Chem. 2019,131,6712–6716 Figure 4. a) Representation and b),c) results from microfluidic determination of the electrophoretic mobilities of native albumin and albumin-2.The device is operated by withdrawingthe sample and flanking buffer from the outlets to create alaminar stream of sample that flows between two electrolyte streams, separated by the buffer. When apotentialdifference is applied between the electrolyte streams, atransverse electric field is created in the separation chamber and the deflection of the sample can be monitored by using the intrinsic fluorescence of the protein at 280 nm.[21] Both samples have aunique response to the electric field and this can be seen by plotting electric field (E)versus the drift velocity (Vdrift)b), which is used to determine the representative mobilities (c). A ngewandte Chemie Communications 6643Angew.Chem. Int.Ed. 2019,58,6640 –6644 T2019 The Authors. Published by Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim www.angewandte.org [1] N. Krall, F. P. da Cruz, O. Boutureira, G. J. L. Bernardes, Nat. Chem. 2015,8,103–113. [2] V. Chudasama, A. Maruani, S. Caddick, Nat. Chem. 2016,8, 114–119. [3] a) C. D. Spicer,B.G.Davis, Nat. Commun. 2014,5,4740;b)O. Boutureira, G. J. L. Bernardes, Chem. Rev. 2015,115,2174 – 2195;c)E.A.Hoyt, P. M. S. D. Cal, B. L. Oliveira, G. J. L. Bernardes, Nat. Rev.Chem. 2019,3,147 –171;d)L.Xue,I.A. Karpenko,J.Hiblot, K. Johnsson, Nat. Chem. Biol. 2015,11, 917–923. [4] a) M. J. Matos,B.L.Oliveira, N. Mart&nez-S#ez, A. Guerreiro, P. M. S. D. Cal, J. Bertoldo,M.Maneiro,E.Perkins,J.Howard, M. J. Deery,J.M.Chalker,F.Corzana, G. Jim8nez-Os8s, G. J. L. Bernardes, J. Am. Chem. Soc. 2018,140,4004–4017;b)S.R. Adusumalli, D. G. Rawale,U.Singh, P. Tripathi, R. Paul, N. Kalra, R. K. Mishra, S. Shukla, V. Rai, J. Am. Chem. Soc. 2018, 140,15114–15123. [5] a) S. Lin, X. Yang,S.Jia, A. M. Weeks,M.Hornsby, P. S. Lee, R. V. Nichiporuk,A.T.Iavarone, J. A. Wells,F.D.Toste,C.J. Chang, Science 2017,355,597 –602;b)M.T.Taylor,J.E.Nelson, M. G. Suero,M.J.Gaunt, Nature 2018,562,563–568. [6] Y. Seki, T. Ishiyama, D. Sasaki, J. Abe,Y.Sohma, K. Oisaki, M. Kanai, J. Am. Chem. Soc. 2016,138,10798–10801. [7] C. B. Rosen, M. B. Francis, Nat. Chem. Biol. 2017,13,697–705. [8] S. Bloom, C. Liu, D. K. Kçlmel, J. X. Qiao,Y.Zhang,M.A.Poss, W. R. Ewing,D.W.C.MacMillan, Nat. Chem. 2017,10,205– 211. [9] a) J. M. Chalker, G. J. L. Bernardes,Y.A.Lin, B. G. Davis, Chem. Asian J. 2009,4,630–640;b)S.B.Gunnoo,A.Madder, ChemBioChem 2016,17,529–553. [10] a) B. Bernardim,P.M.S.D.Cal, M. J. Matos,B.L.Oliveira, N. Mart&nez-S#ez, I. S. Albuquerque,E.Perkins,F.Corzana, A. C. B. Burtoloso,G.Jim8nez-Os8s, G. J. L. Bernardes, Nat. Commun. 2016,7,13128;b)B.Bernardim, M. J. Matos,X. Ferhati, I. CompaÇln, A. Guerreiro,P.Akkapeddi, A. C. B. Burtoloso,G.Jim8nez-Os8s, F. Corzana, G. J. L. Bernardes, Nat. Protoc. 2019,14,86–99. [11] E. V. Vinogradova, C. Zhang,A.M.Spokoyny,B.L.Pentelute, S. L. Buchwald, Nature 2015,526,687 –691. [12] C. Zhang,M.Welborn, T. Zhu, N. J. Yang,M.S.Santos,T. VanVoorhis,B.L.Pentelute, Nat. Chem. 2015,8,120–128. [13] a) G. J. L. Bernardes,J.M.Chalker, J. C. Errey,B.G.Davis, J. Am. Chem. Soc. 2008,130,5052 –5053;b)A.M.Freedy,M.J. Matos,O.Boutureira, F. Corzana, A. Guerreiro,P.Akkapeddi, V. J. Somovilla, T. Rodrigues,K.Nicholls,B.Xie,G.Jim8nezOs8s, K. M. Brindle,A.A.Neves,G.J.L.Bernardes, J. Am. Chem. Soc. 2017,139,18365–18375. [14] C. Zhang,P.Dai, A. A. Vinogradov,Z.P.Gates,B.L.Pentelute, Angew.Chem. Int. Ed. 2018,57,6459–6463; Angew.Chem. 2018,130,6569–6573. [15] J. M. J. M. Ravasco,H.Faustino,A.Trindade,P.M.P.Gois, Chem. Eur.J.2019,25,43–59. [16] F.-H. Ma, J.-L. Chen, Q.-F.Li, H.-H. Zuo,F.Huang,X.-C.Su, Chem. Asian J. 2014,9,1808 –1816. [17] G. M. Dubowchik, R. A. Firestone, Bioorg.Med. Chem. Lett. 1998,8,3341–3346. [18] P. L. Carl, P. K. Chakravarty,J.A.Katzenellenbogen, J. Med. Chem. 1981,24,479 –480. [19] J. R. Junutula, H. Raab,S.Clark, S. Bhakta, D. D. Leipold, S. Weir, Y. Chen, M. Simpson,S.P.Tsai, M. S. Dennis,Y.Lu, Y. G. Meng,C.Ng, J. Yang,C.C.Lee,E.Duenas,J.Gorrell, V. Katta, A. Kim, K. McDorman, K. Flagella,R.Venook, S. Ross,S.D. Spencer, W. Lee Wong, H. B. Lowman, R. Vandlen, M. X. Sliwkowski, R. H. Scheller,P.Polakis,W.Mallet, Nat. Biotechnol. 2008,26,925. [20] K. L. Saar,Y.Zhang,T.Mgller,C.P.Kumar,S.Devenish, A. Lynn, U. Łapin ´ska, X. Yang,S.Linse,T.P.J.Knowles, Lab Chip 2018,18,162–170. [21] P. K. Challa, Q. Peter,M.A.Wright, Y. Zhang,K.L.Saar,J.A. Carozza,J.L.P.Benesch, T. P. J. Knowles, Anal. Chem. 2018,90, 3849–3855. [22] M. Forster,A.Chaikuad, T. Dimitrov,E.Dçring,J.Holstein, B. T. Berger,M.Gehringer, K. Ghoreschi, S. Mgller,S.Knapp, S. A. Laufer, J. Med. Chem. 2018,61,5350–5366. [23] E. Weerapana, C. Wang,G.M.Simon, F. Richter,S.Khare, M. B. D. Dillon, D. A. Bachovchin, K. Mowen, D. Baker,B.F. Cravatt, Nature 2010,468,790–795. Manuscript received:January 31, 2019 Revised manuscript received: March 11, 2019 Acceptedmanuscript online: March 21, 2019 Version of record online: April 9, 2019 A ngewandte Chemie Communications 6644 www.angewandte.org T2019 The Authors. Published by Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim Angew.Chem.Int. Ed. 2019,58,6640 –6644