25 L E Bell Science 321 1457ndash1461 (2008)26 R M Silverstein G C Bassler T C Morrill Spectrometric

Identification of Organic Compounds (John Wiley and Sonsed 4 1981)

27 B H Stuart Infrared Spectroscopy Fundamentals andApplications (John Wiley amp Sons 2005)

28 J V Gulmine P R Janissek H M Heise L Akcelrud PolymTest 21 557ndash563 (2002)

29 V Liu S Fan Comput Phys Commun 183 2233ndash2244(2012)

30 X Huang J Solid State Electrochem 15 649ndash662(2011)

31 H L Zhu Z Q Fang C Preston Y Y Li L B Hu EnergEnviron Sci 7 269ndash287 (2014)

32 V Mrstina F Fejgl Needle Punching Textile Technology TextileScience and Technology (Elsevier 1990)

33 H Lee S M Dellatore W M Miller P B Messersmith Science318 426ndash430 (2007)

34 M H Ryou Y M Lee J K Park J W Choi Adv Mater 233066ndash3070 (2011)

35 X Liu et al ACS Nano 7 9384ndash9395 (2013)36 J Jiang et al ACS Appl Mater Interfaces 5 12895ndash12904

(2013)37 Z-Y Xi Y-Y Xu L-P Zhu Y Wang B-K Zhu J Membr Sci

327 244ndash253 (2009)

ACKNOWLEDGMENTS

This work was sponsored by the Advanced Research ProjectsAgencyndashEnergy (ARPA-E) US Department of Energy underaward DE-AR0000533 The authors thank H Dai for lending thethermal camera All data are available in the manuscript and thesupplementary materials YC SF P-CH AYS PBC andYP have a US patent application (no 62296549) related to thiswork YC and P-CH conceived the idea P-CH and AYSconducted the FTIR spectrometry measurement P-CH conductedUV-Vis spectrometry measurement P-CH and CL designed

and conducted the thermal measurement P-CH AYS andJX analyzed the FTIR data AYS performed the RCWA simulationP-CH and PBC constructed and simulated heat transportmodel P-CH conducted SEM and optical microscopecharacterization P-CH and YP performed wearability testsYC and SF supervised the project All authors contributed tothe writing of the paper

SUPPLEMENTARY MATERIALS

wwwsciencemagorgcontent35363031019supplDC1Materials and MethodsSupplementary TextFigs S1 to S8Table S1References (38ndash42)

25 February 2016 accepted 21 July 2016101126scienceaaf5471

ASYMMETRIC CATALYSIS

Ligand-accelerated enantioselectivemethylene C(sp3)ndashH bond activationGang Chen1 Wei Gong1 Zhe Zhuang1 Michal S Andrauml1 Yan-Qiao Chen1 Xin Hong2

Yun-Fang Yang2 Tao Liu1 K N Houk2dagger Jin-Quan Yu1dagger

Effective differentiation of prochiral carbonndashhydrogen (CndashH) bonds on a single methylenecarbon via asymmetric metal insertion remains a challenge Here we report the discovery ofchiral acetyl-protected aminoethyl quinoline ligands that enable asymmetric palladiuminsertion into prochiral CndashH bonds on a single methylene carbon centerWe apply thesepalladium complexes to catalytic enantioselective functionalization of b-methylene CndashH bondsin aliphatic amides Using bidentate ligands to accelerate CndashH activation of otherwiseunreactivemonodentate substrates is crucial for outcompeting the background reaction drivenby substrate-directed cyclopalladation thereby avoiding erosion of enantioselectivityThepotential of ligand acceleration in CndashH activation is also demonstrated by enantioselectiveb-CndashH arylation of simple carboxylic acids without installing directing groups

Enantioselective functionalization of pro-chiral CndashH bonds can potentially lead toa broad range of efficient routes to chiralcompounds Despite extensive efforts thescope and efficiency of enantioselective

C(sp3)ndashH activation reactions are far from ade-quate for broad applications in asymmetric syn-thesis (1 2) Enantioselective carbene andnitreneinsertions into C(sp3)ndashH bonds have been dem-onstrated in both diastereoselective and enantio-selective fashion (3ndash7) However asymmetricC(sp3)ndashH activation reactions via metal insertionare limited to thedesymmetrizationof twoprochiralcarbon centers (8ndash15) (Fig 1A) For example de-symmetrizations of relatively reactive cyclopropyland cyclobutyl CndashHbonds have been achievedwithPd(II) catalysts and chiral monoprotected aminoacid (MPAA) ligands (8ndash11) Desymmetrizationof two carbon centers has also been achieved

through a Pd(0)-catalyzed intramolecular CndashHarylation as demonstrated in a series of pioneeringstudies (12ndash15) Thus far development of an ef-ficient chiral metal catalyst that can differentiateprochiral CndashHbonds residingona singlemethylenecarbon center via metal insertion remains a chal-lenge In terms of synthetic disconnection such aprocess is also distinct from the desymmetriza-tion as the newly created chiral center of amidesubstrates resides at the b-methylene carbon in-stead of the a-carbon center Recently a transientchiral directing group has also been shown toperformenantioselective CndashHarylation of benzylicCndashHbonds (16) However the transient amino aciddirecting group does not promote alkyl methyleneCndashH activation Furthermore the transient di-recting group is also incompatible with sub-strates derived from carboxylic acidsThe use of a bidentate 8-aminoquinoline di-

recting group and a chiral phosphoric amide lig-and has affordedmoderate enantiomeric ratios(er) ranging from 7426 to 919 with benzyl CndashHbonds though this method is much less suc-cessful with alkyl CndashH bonds (6337 er) (17) Ingeneral such strongly coordinating directinggroups promote ligandless CndashH activation re-actions which could be detrimental for asymmetric

catalysis as these background reactions erodeenantioselectivity Bidentate coordination fromsubstrates also prevents the exploitation of a widerange of potentially powerful chiral bidentate lig-ands in palladium catalysis due to a lack of vacantcoordination sites Practically the requirement forbidentate coordination from substrates precludestheuseof a variety of simplemonodentatedirectinggroups and native functional groups to directCndashH activation an important goal of the fieldDespite the aforementioned challenges enantio-

selective b-CndashH functionalization has long beenthe focus of our research efforts due to the im-portance of constructing b-chiral centers inasymmetric synthesis Current retrosynthetic dis-connections for the asymmetric synthesis ofb-functionalized chiral carboxylic acids or amidesextensively use conjugate addition reactions of thecorresponding olefins Rh(I)-catalyzed asymmetricconjugate addition of ab-unsaturated ketoneswith aryl boronic acids has afforded a usefulmethod for the preparation of chiral b-arylatedcompounds (18 19) However when a given sub-strate or synthetic intermediate contains a sat-urated aliphatic acid chain without double bondsdirect enantioselective arylation of the methyleneCndashH bonds at the b position of amides throughpalladium insertion provides a solution (Fig 1B)In our early efforts we adopted a chiral auxiliaryapproach to gain insight into stereoselective pal-ladium insertion into b-C (sp3)ndashHbonds (20) How-ever development of an enantioselective versionof these diastereoselective b-CndashH iodination andacetoxylation reactions has not been successfulowing to the lack of an appropriate ligand thatcan match the strongly coordinating oxazolinedirecting group (21) Employing a weakly coordi-nating amide directing group in combination withchiralMPAA ligands has led to desymmetrizationof methyl cyclopropyl and cyclobutyl CndashHbonds(Fig 1A) at two different carbon centers (9 10)Unfortunately MPAA ligands have proven in-effective in promoting palladium insertion intoacyclic methylene CndashH bondsHere we report the discovery of chiral acetyl-

protected aminoethyl quinoline (APAQ) ligandsthat enable Pd(II)-catalyzed enantioselective aryla-tionof b-methyleneCndashHbonds of aliphatic amideswith enantiomeric ratios reaching up to 964 and

SCIENCE sciencemagorg 2 SEPTEMBER 2016 bull VOL 353 ISSUE 6303 1023

1The Scripps Research Institute 10550 North Torrey PinesRoad La Jolla CA 92037 USA 2Department of Chemistryand Biochemistry University of California Los Angeles CA90095-1569 USAThese authors contributed equally to this work daggerCorrespondingauthor Email houkchemuclaedu (KNH) yu200scrippsedu(J-QY)

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yields as high as 89 (Fig 1C) These APAQ ligandscontaining quinoline and acetyl-protected aminocoordinating moieties form six-membered bis-chelating rings with palladium which drastically

acceleratesmethylene CndashHactivation thereby con-trolling the stereoselectivity In contrast the acetyl-protected aminomethyl quinoline coordinatingwith Pd(II) via five-membered bis-chelation is

completely inactive in this reaction Concep-tually the combination of weakly coordinatingmonodentate substrates and ligand accelerationopens the possibility for using a variety of simple

1024 2 SEPTEMBER 2016 bull VOL 353 ISSUE 6303 sciencemagorg SCIENCE

Fig 1 Enantioselective meth-ylene CndashH activation reac-tions (A) Enantioselective CndashHactivation via desymmetrizationof two carbon centers (B) Twosynthetic disconnections(C) Differentiating prochiral CndashHbond on a single methylenecarbon center DG directinggroup PG protecting group OTftrifluoromethanesulfonate Araryl group Ac acetyl group Etethyl group Bu butyl group Ralkyl or aryl group

∆∆

∆∆

Fig 2 Ligand optimization for enantioselective methylene CndashH arylation (A) Yields were determined by 1H nuclear magnetic resonance analysis of thecrude product using CH2Br2 as an internal standard Enantiomeric ratios were determined by chiral high-performance liquid chromatography The absoluteconfiguration of L35was determined by x-ray crystallography (fig S4) p-Tol-I para-tolyl iodide equiv equivalent HFIP hexafluoro-2-propanol Memethyl groupPh phenyl group Pr propyl group Bn benzyl group NR no reaction The asterisk indicates the presence of a chiral center at the atom (B) DFT-optimizedstructures and relative free energies of the two enantiomeric CndashH metalation-deprotonation transition states

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coordinating groups including native functionalgroups to direct enantioselective CndashH activationas demonstratedwith free carboxylic acid substratesGuided by our overarching goal of developing

ligand-accelerated enantioselective CndashHactivationof weakly coordinating substrates we set out touse the electron-deficient amide substrate 1 andevaluate the effects of chiral ligands on the ex-tensively studied CndashH arylation reaction (22ndash24)Following our previous finding that quinoline andpyridine ligands can accelerate C(sp3)ndashH activation(25 26) we prepared a number of correspondingchiral ligands (including L4 and L5) and exam-ined their activity under standard reaction con-ditions (table S1)Unfortunately thesemonodentatechiral ligands do not exert substantial influenceon the stereochemistry of the palladium insertionstep Considering the effectiveness of bidentateMPAA ligands in controlling the stereochemistryof Pd-catalyzed desymmetrization of prochiralcyclopropyl and cyclobutyl CndashH bonds on twocarbon centers we began to develop bidentateligands incorporating structuralmotifs frombothquinoline and MPAA ligands The crucial role ofthe NHAc (Ac acetyl) moiety ofMPAA ligands inthe CndashHcleavage step identified by experimental(8 27) and computational studies (28 29) promp-ted us to develop acetyl-protected aminomethylquinoline ligands that incorporate this coordinat-ing moiety Disappointingly such ligands (L6to L8) resulted in a complete loss of reactivity(Fig 2A and table S1) We reasoned that the five-membered bidentate chelation with Pd(II) could

result in the formation of a stable but inactivepalladium complex tetra-coordinated with twoligands As such we prepared APAQ and amino-propyl quinoline ligands (L9 andL10) that wouldcoordinatewithPd(II) via six- and seven-memberedchelate structures respectively both of whichshould havemarkedly reduced binding constantscompared to the corresponding five-memberedchelate (L6) Such subtlemodification restored thereactivity with L9 and L10 thus offering a bi-dentate ligand scaffold for further developmentAlthough aminopropyl quinoline L10 is more

reactive than aminoethyl quinolineL9 we choseto focus on the latter scaffold because of itssynthetic accessibility We used Ellmanrsquos highlyefficient asymmetric imine addition reaction (30)to prepare a series of chiral APAQ ligands from2-methylquinoline and optically pure sulfinylimines We initially found that ligand L11 (tableS1) containing an a-methyl group at the chiralcenter enhanced the reactivity considerably (75yield) albeit with poor enantioselectivity (4753 er)The a-methyl group was then replaced with var-ious alkyl groups Only the sterically bulky iso-propyl groupwas found to produce a substantiallyimproved enantiomeric ratio (2773) but it alsoled to diminished yield (L16 table S1) Althoughfurther tuning of the alkyl substitution provedless promising the result obtained with thea-phenyl substitution in L17 provided us withan encouraging lead for ligand optimization (76yield 2971 er) With L17 in hand we examinedthe effect of the protecting groups on the amino

group (table S2) Replacing the acetyl-protectinggroup by more sterically hindered analogousmotifs decreased the yields considerably Othertypes of protecting groups such as carbamatesand sulfonyls proved completely inactive Wethus prepared a number of APAQ ligands [L18to L33 (table S1)] with a range of steric and elec-tronic variation on the a-phenyl ring We foundthat the steric effect is predominant as indicatedby the markedly improved yield and enantio-selectivity obtained with ligand L32 bearingthe sterically hindered 35-di-tert-butylphenyl group(85 yield 1981 er) At this point of optimizationwe introduced a second chiral center at the benzylicposition hoping to further improve the enantio-selectivity Attributing the origin of the stereo-selectivity to differentially hindered faces on thesquare planar palladium complex (8ndash11) we fo-cused on the variations of syn-APAQ ligands inwhich both substituentswould point up- or down-ward upon chelating with Pd(II) The introduc-tion of a methyl group at the benzylic position(L34) afforded a substantial improvement inenantioselectivity (9010 er) while maintainingthe high yield A slightly more bulky ethyl group(L35) further improved the enantioselectivity to92575 er Further increasing steric hindrance atthe benzylic position decreased both yield andenantioselectivity (L36 to L39) To obtain in-sight into the stereochemicalmodel of this enantio-selective palladium-insertion process we alsotested the anti-APAQ ligands (L40 and L41)Although both yield and enantioselectivity droppedconsiderably with these two anti-ligands thereversal of chiral induction by altering the ab-solute configuration at thea position suggests thatthe chiral center adjacent to the amino groupdictates enantioselection (table S3)Building on results from previous computa-

tional studies (28 29) we explored the enantio-selectivity in the CndashH metalation-deprotonationstep with density functional theory (DFT) usingligand L34 and substrate 1 The optimized struc-tures and relative free energies of the most fa-vorable transition states for the two enantiomersare shown in Fig 2B In both transition statesthe palladium(II) center is coordinated with thequinoline nitrogen and the amide nitrogen ofthe deprotonated APAQ ligand as well as onenitrogen from the amide substrate formed bydeprotonation The oxygen of the amide of theAPAQ ligand acts as an intramolecular base todeprotonate the methylene CndashH and facilitatethe PdndashC bond formation TS_R is more fa-vorable than TS_S by 12 kcalmol in excellentagreement with the experimental selectivity(9010 er) The distinct six-membered bis-chelatedesign and the bulky a-aryl substituent orient theamide in such a way that the b-CndashH bond is ac-tivated The terminalmethyl groupof the substrateis oriented differently in the R and S transitionstates with more severe substrate distortion inTS_S where the methyl group of the substratemethylene is on the same face as the bulky a-arylsubstituent of the ligand and the peri-hydrogenof the quinoline This discrepancy underlies theenantioselectivity (see supplementarymaterials)

SCIENCE sciencemagorg 2 SEPTEMBER 2016 bull VOL 353 ISSUE 6303 1025

Fig 3 Scope of aryl iodides in enantioselective methylene CndashH arylation Data are reported asisolated yield of purified compound The absolute configurations of 2a 2e and 2k were determinedby x-ray crystallography (figs S5 to S7)

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With the optimal ligand L35 in hand wefurther optimized the reaction conditions for thearylation of 1 and improved the enantioselectivityto 955 er (table S4 entry 21) We next surveyedthe scope of aryl iodides for this enantioselectiveb-CndashH arylation (Fig 3) Arylation of 1 with sim-ple iodobenzene afforded the desired product in89 yield and 955 er (2b) A wide range of para-substituted aryl iodideswas employed as couplingpartners Electron-donating groups afforded excel-lent enantioselectivity and good yields (2a 2cand 2d) Electron-deficient aryl iodides bearingtrifluoromethoxy fluoro chloro bromo ketoneand ester substituents were also compatible pro-viding consistently high enantioselectivity (2e to2h 2j 2k) although the yield dropped to 45with trifluoromethyl substitution (2i) Similarresults were obtained with metasubstituted aryliodides (2l to2p) Although slightly lower enantio-selectivitywasobtainedwithortho-methoxylphenyliodide (2q) the ortho-ester group was well tol-erated (2r) An aryl iodide containing phospho-natemoiety also afforded synthetically useful yieldand enantioselectivity (2s) Disubstituted aryliodides alsoproved tobe suitable couplingpartners(2t to 2v)

This protocol for enantioselective arylationofmethylene CndashHbonds is also applicable to otheraliphatic amides (Fig 4) Aliphatic amides withvarious chain lengthswerewell tolerated providingproducts 4a to 4dwith excellent enantioselectivityand in high yields The direct enantioselectiveb-CndashH arylation of amides derived fromnaturallyoccurring caprylic acid and myristic acid demon-strates the utility of this method when a doublebond is not available for conjugate addition (4cand 4d) Substrates containing sterically hinderedalkyl groups at the b position (cyclopentyl clyclo-hexyl) provided good enantioselectivity but loweryields (4e and 4f) Isopropyl cyclopentyl cyclo-hexyl and cyclohexylmethyl moieties at the g po-sitions were well tolerated affording satisfactoryyields and enantioselectivity (4g to 4j) Phenylester amino ether and ketone functionalities atthe d and e positions consistently afforded highenantioselectivity (4k to 4p) However loweryields were obtained with the ether and ketonesubstrates (4o and4p) Piperidine at the g positionafforded good yield and high enantioselectivity(4q) whereas the presence of piperidine at theb position gave lower yield (4r) The presence ofa tetrahydropyran motif at the g position was

also well tolerated affording synthetically usefulyield and enantioselectivity (4s) The enolate ofthe corresponding ester of product 4s reactedwith di-tert-butyl azodicarboxylate to give thechiral a-amino ester in gt201 diastereoselectivityproviding a route for the preparation of complexchiral amino acids (see supplementary materials)Arylation of benzylic CndashH with 3t using ligandL35 provided poor yield and enantioselectivity(38 yield 6832 er) Switching to ligand L32resulted in a marked improvement in both yieldand enantioselectivity (4t) b-phenyl groups con-taining both electron-withdrawing and -donatinggroups were also compatible with this reaction(4u and 4v) To demonstrate the advantage ofusing weakly coordinating monodentate sub-strates and chiral bidentate ligands we appliedthe enantioselective b-CndashH arylation protocol totwo free carboxylic acids to provide 4w and 4x ascis-diastereomers exclusively These products arenot accessible by asymmetric conjugate additionIn summary a chiral bidentate APAQ ligand

scaffold was found to enable Pd-catalyzed enantio-selective arylation of prochiral b-CndashH bonds on asingle methylene carbon center The feasibility ofasymmetric palladium insertion into ubiquitousmethylene CndashH bonds with monodentate sub-strates opens a new avenue for developing awiderange of synthetically useful enantioselectiveCndashH activation reactions

REFERENCES AND NOTES

1 R Giri B-F Shi K M Engle N Maugel J-Q Yu Chem SocRev 38 3242ndash3272 (2009)

2 D A Colby A S Tsai R G Bergman J A Ellman Acc ChemRes 45 814ndash825 (2012)

3 M P Doyle R Duffy M Ratnikov L Zhou Chem Rev 110704ndash724 (2010)

4 R P Reddy H M L Davies Org Lett 8 5013ndash5016(2006)

5 C Liang et al Angew Chem Int Ed 45 4641ndash4644(2006)

6 D N Zalatan J Du Bois J Am Chem Soc 130 9220ndash9221(2008)

7 E Milczek N Boudet S Blakey Angew Chem Int Ed 476825ndash6828 (2008)

8 B-F Shi N Maugel Y-H Zhang J-Q Yu Angew Chem IntEd 47 4882ndash4886 (2008)

9 M Wasa K M Engle D W Lin E J Yoo J-Q Yu J AmChem Soc 133 19598ndash19601 (2011)

10 K-J Xiao et al J Am Chem Soc 136 8138ndash8142 (2014)11 K S L Chan H-Y Fu J-Q Yu J Am Chem Soc 137

2042ndash2046 (2015)12 M Nakanishi D Katayev C Besnard E P Kuumlndig Angew

Chem Int Ed 50 7438ndash7441 (2011)13 S Anas A Cordi H B Kagan Chem Commun (Camb) 47

11483ndash11485 (2011)14 N Martin C Pierre M Davi R Jazzar O Baudoin Chemistry

18 4480ndash4484 (2012)15 T Saget S J Lemouzy N Cramer Angew Chem Int Ed 51

2238ndash2242 (2012)16 F-L Zhang K Hong T-J Li H Park J-Q Yu Science 351

252ndash256 (2016)17 S-B Yan S Zhang W-L Duan Org Lett 17 2458ndash2461

(2015)18 G Berthon T Hayashi in Catalytic Asymmetric Conjugate

Reactions A Cordova Ed (Wiley 2010) pp 1ndash6719 J-F Paquin C Defieber C R J Stephenson E M Carreira

J Am Chem Soc 127 10850ndash10851 (2005)20 R Giri X Chen J-Q Yu Angew Chem Int Ed 44

2112ndash2115 (2005)21 K M Engle J-Q Yu J Org Chem 78 8927ndash8955

(2013)22 V G Zaitsev D Shabashov O Daugulis J Am Chem Soc

127 13154ndash13155 (2005)

1026 2 SEPTEMBER 2016 bull VOL 353 ISSUE 6303 sciencemagorg SCIENCE

Fig 4 Scope of amides and acids in enantioselective methylene CndashH arylation Data are reportedas isolated yield of purified compound Asterisks in the structures indicate the presence of a chiral centerat the atom The symbol following a compound number indicates 15 equiv of Ag2CO3 25 equiv aryliodide and 12 mole (mol ) L32 The absolute configuration of 4u was determined by x-ray crystal-lography (fig S8)The dagger symbol indicates 10 equiv K2HPO4 as additive 20mol L16 and 100degCTheDagger symbol indicates 10 equiv Na2HPO47H2Oas additive 20 equiv AgOAc 20 equiv aryl iodide and 12molL32The sect symbol indicates that yields and er valueswere determined fromcorrespondingmethyl esters (seesupplementary materials for experimental details) Phth phthalimido group Ts tosyl group

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23 B V S Reddy L R Reddy E J Corey Org Lett 83391ndash3394 (2006)

24 Y Feng G Chen Angew Chem Int Ed 49 958ndash961 (2010)25 M Wasa et al J Am Chem Soc 134 18570ndash18572 (2012)26 J He et al Angew Chem Int Ed 55 785ndash789 (2016)27 K S L Chan et al Nat Chem 6 146ndash150 (2014)28 D G Musaev A Kaledin B-F Shi J-Q Yu J Am Chem Soc

134 1690ndash1698 (2012)29 G-J Cheng et al J Am Chem Soc 136 894ndash897 (2014)30 M T Robak M A Herbage J A Ellman Chem Rev 110

3600ndash3740 (2010)

ACKNOWLEDGMENTS

We thank The Scripps Research Institute and the NIH (NationalInstitute of General Medical Sciences grant 2R01GM084019) forfinancial support We also thank the Shanghai Institute of Organic

Chemistry Zhejiang Medicine and Pharmaron for a postdoctoralfellowship (GC) and the Deutsche Forschungsgemeinschaft for aresearch fellowship (MSA) We acknowledge earlier computationalstudies that enhanced our understanding of catalyst developmentconducted in collaboration with J Musaev and K Houk within theNSF Center for Chemical Innovation Center for Selective CndashHFunctionalization (grant CHE-1205646) Author contributionsJ-QY conceived the concept GC and WG developed the chiralligands and optimized the reaction conditions ZZ MSA Y-QC and TL optimized the chiral ligands and surveyed thesubstrate scope XH Y-FY and KNH performed the DFTcalculation J-QY directed the project J-QY and The ScrippsResearch Institute have filed a provisional patent application(62311039) Metrical parameters for the structures of L13 L21L40b L35 2a 2e 2k and 4u (see supplementary materials)are available free of charge from the Cambridge Crystallographic

Data Centre under reference number CCDC-14522020 CCDC-14522021 CCDC-14522023 CCDC-14522022 CCDC-14522016CCDC-14522017 CCDC-14522018 and CCDC-14522019 respectively

SUPPLEMENTARY MATERIALSwwwsciencemagorgcontent35363031023supplDC1Materials and MethodsFigs S1 to S9Tables S1 to S14NMR SpectraHPLC TracesReferences (31ndash55)

9 February 2016 resubmitted 4 April 2016accepted 9 August 2016101126scienceaaf4434

PLANT SCIENCE

Plant development regulated bycytokinin sinksEvelyne Zuumlrcher1 Jingchun Liu1 Martin di Donato2 Markus Geisler2 Bruno Muumlller1dagger

Morphogenetic signals control the patterning of multicellular organisms Cytokinins aremobile signals that are perceived by subsets of plant cellsWe found that the responses tocytokinin signaling during Arabidopsis development are constrained by the transporterPURINE PERMEASE 14 (PUP14) In our experiments the expression of PUP14 was inverselycorrelated to the cytokinin signaling readout Loss of PUP14 function allowed ectopiccytokinin signaling accompanied by aberrant morphogenesis in embryos roots and theshoot apical meristem PUP14 protein localized to the plasma membrane and importedbioactive cytokinins thus depleting apoplastic cytokinin pools and inhibiting perception byplasma membranendashlocalized cytokinin sensors to create a sink for active ligandsWepropose that the spatiotemporal cytokinin sink patterns established by PUP14 determinethe cytokinin signaling landscape that shapes the morphogenesis of land plants

Multicellular organisms depend on differ-ential cell functions controlled by signalingsystems The precise determination ofsignal-perceiving cells is important toensure normal development Cytokinins

are chemical plant signals that control morpho-genesis integrate environmental cues andmediatebiotic interactions (1ndash3) Cytokinins are perceivedby largely redundantly acting hybrid kinases thatactivate a phosphorelay circuitry to stimulate thetranscription of target genes The spatiotemporalprecision of the signaling patterns in different plantorgans (4 5) raises the question of how controlis establishedEach step involved in eliciting a signaling re-

sponse including ligandbiosynthesis and expressionof signaling components could be differentiallyregulated and contribute to defining the signalingpatterns to a greater or lesser degree To identifylimiting and regulated steps we used Arabidop-sis heart-stage embryos as a model in which thecytokinin response marks the provascular tis-sue (Fig 1A) First to evaluate whether bioactive

cytokinins are limited we incubated embryosfor 16 hours with the degradation-insensitivecytokinin benzyladenine (BA) (6) This caused astereotypic expansion of the synthetic cyto-kinin reporter TCSnGFP (Two Component sig-naling Sensor newgreen fluorescent protein) (5)(Fig 1A) thereby confirming that cytokinin lev-els are controlled (7) However excess cytokininsdid not induce TCSnGFP expression in the pros-pective cotyledons despite the transcription ofthe cognate cytokinin receptor ARABIDOPSISHISTIDINE KINASE 4 (AHK4) in these domains(Fig 1A) this finding suggests that failure to turnon signaling cannot be explained by missing re-ceptors To test whether signaling downstreamof receptors is functional we expressed CYTO-KININ INDEPENDENT 1 (CKI1) CKI1 encodesa hybrid kinase with cytokinin-independent con-stitutive activity (3) Its short-term expressioncaused ubiquitous TCSnGFP activation (Fig 1A)Together these results suggest that cells of theprospective cotyledons fail to activate cytokininsignaling despite a functional signaling systemand even upon addition of abundant active ligandWe hypothesized that productive ligand-receptor

interactions within organs could depend on cyto-kinin transporters that guide differential cellularlocalization of cytokinins To test whether mem-bers of the Arabidopsis PURINE PERMEASE

(PUP) family of transmembrane proteins impli-cated in cytokinin translocation (8) control thespatiotemporal landscape of cytokinin signalingwe first established a transcription profile of allfamily members based on our own analysis andpublished transcriptome data (9ndash11) (fig S1) PUP14expression was unique in that it prevailed in allorgans and stages analyzed including embryos Todetermine the PUP14 expression pattern we ana-lyzedPUP14PUP14-GFP transgenic plants Inheart-stage embryos PUP14-GFP localized to cells thatfailed to respond to cytokinins including cells ofthe prospective cotyledons (Fig 1B)We confirmedthis pattern by mRNA in situ hybridization with aPUP14 antisense probe (Fig 1B and fig S2A) Theexclusive nature of PUP14 expression and the cyto-kinin signaling pattern are compatiblewith an inhi-bitory function of PUP14 in the cytokinin responseTo eliminate PUP14 function during defined

timewindows thereby avoiding secondary effectsand potential lethality issues we constructed anethanol-inducible artificial microRNA (amiR) (12)targeting PUP14 (35SgtALCgtamiRPUP14) Uponinduction of the amiRPUP14 transgene PUP14mRNAandPUP14-GFP levels were reducedwithin24 hours (Fig 1D and fig S2 B and C) InducingamiRPUP14 expression for 16 hours caused wide-spread ectopic cytokinin signaling in the embryo(Fig 1B) even in cells of the prospective cotyledonsthat are nonresponsive to treatments with exoge-nous cytokinins (Fig 1A) this result supports a roleof PUP14 in confining the cytokinin response Thesame treatment regime did not affect the auxinresponse (fig S2D) indicating that PUP14 actsspecifically on cytokinin signaling After 2 days ofamiRPUP14 induction morphological defectsin the prospective cotyledons and the nascentroot meristem became apparent (Fig 1C) con-sistent with the ectopic cytokinin responses inthese domains The amiRPUP14-induced pheno-types were complemented by an amiRPUP14-resistant transgene (PUP14) encompassing thePUP14 locus (fig S2 E toG) which suggests thatthe inducible amiRPUP14 acts specifically In ad-dition an inducible amiR against nonexpressedPUP19 and PUP20 (fig S1) did not cause obviousphenotypes (fig S2 E to G) Finally a T-DNA in-sertion to thePUP14promoter causing a reductionin PUP14mRNA levels showed qualitatively sim-ilar but weaker phenotypes in embryos seedlingsand adult shoots relative to amiRPUP14-induced

SCIENCE sciencemagorg 2 SEPTEMBER 2016 bull VOL 353 ISSUE 6303 1027

SUPPLEMENTARY MATERIALS

1Zuumlrich-Basel Plant Science Center Department of Plant andMicrobial Biology University of Zuumlrich 8008 ZuumlrichSwitzerland 2Plant Biology Department of BiologyUniversity of Fribourg 1700 Fribourg SwitzerlandThese authors contributed equally to this work daggerCorrespondingauthor Email brunomuelleruzhch

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H bond activationminus)3Ligand-accelerated enantioselective methylene C(sp

Jin-Quan YuGang Chen Wei Gong Zhe Zhuang Michal S Andrauml Yan-Qiao Chen Xin Hong Yun-Fang Yang Tao Liu K N Houk and

DOI 101126scienceaaf4434 (6303) 1023-1027353Science

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