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Vial, Chem. Commun., 2015, DOI: 10.1039/C5CC07628E.

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Institut des Biomolécules Max Mousseron, UMR 5247 CNRS – Université deMontpellier,PlaceEugèneBataillon,cc1706,34296Montpelliercedex5,France.E-mail:laurent.vial@univ-montp2.fr† Electronic Supplementary Information (ESI) available: synthetic procedure forD3CF; details for the analytical and computational methods. SeeDOI:10.1039/x0xx00000x

:Received00thJanuary20xx,Accepted00thJanuary20xx

DOI:10.1039/x0xx00000x

www.rsc.org/

AKISS(KeepItSimple,Sensor)ArrayforGlycosaminoglycans†Jean-PatrickFrancoiaandLaurentVial*

Herein,wedemonstratethatitispossibletodesignasensorarraywithonemultivalentcationicreceptor(adendrigraftoflysine)andone fluorescent anionic indicator (a fluorescein-labeled peptide).Depending on the loading of the indicator on the receptor,negatively charged glycosaminoglycans (GAGs) induce a positiveornegativevariationofthefluorescentsignalastheydisplacetheindicatorsfromthereceptorortheycompacttheindicatorsonthereceptor’s surface, respectively. This unique strategy allows notonlytheblindidentificationofpureGAGswithalevelofaccuracyof100%,butalsothedifferentiationofmixtures.

Glycosaminoglycans (GAGs) are an important class ofexogenous biopolymers that display numerous therapeuticactivities.1 Because GAGs are structurally analogous (Fig. 1),orthogonalandlengthyanalyticaltechniques–involvingNMRspectroscopy, HPLC, and capillary electrophoresis – arenecessary to identify them.2 The ability to routinelydiscriminate and assess the purity of these anionicpolysaccharidesismandatoryinordertopreventpublichealthdisasters.Indeed,theuseofcontaminatedheparininpatientsundergoing dialysis resulted in serious acute hypersensitivityreactions (including some resulting in death) during the late2000s.3Contaminantsinadulteratedlotsincludedchondroitinsulfate A, dermatan sulfate (chondroitin sulfate B), andhyaluronic acid. To address this issue, some research groupsdevelopedpattern-based recognitionassays for variousGAGsby using arrays of nanoparticles,macrocycles, or liposomes.4Despite the successof thoseworks,extensive synthetic stepswere systematically involved toobtain themultiple receptorsand/orindicatorsthatwerenecessarytoconstructthearrays;therefore impeding their use for cheap routine screening ofGAGs. Herein amuch simpler sensor array for GAGs, from asinglereceptorandasingleindicator,ispresented.

Fig.1Left, from top tobottom:major repeatunitof selectedGAGs.Top right:bioconjugate between a homopolymer of aspartic acid and 5(6)-carboxyfluorescein, D3CF.Bottom right: minimized structurewith electrostaticpotential surfaceof the third-generationdendrigraftpoly-L-lysinepolymerDGLG3.

We recently reported that a cationic dendrigraft poly-L-lysinepolymer(DGL)wasabletoformamulti-ligandcomplexwithaconjugatebetweenananionichomopolymerofasparticacid and 5(6)-carboxyfluorescein; leading to the extinction oftheopticalsignalasaresultofitsaggregationonthesurfaceofthe receptor.5 Fluorescence could be restored upon theintroductionofheparininanindicator-displacementprocess.6Although this sensing ensemble allowed us to detect and

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quantify–forthefirsttime–heparininhumanbloodsamplesatclinicallyrelevant levels, itwasnotpossibletodiscriminatebetweenGAGssuchasheparinandchondroitinsulfate.Inthispreviousstudy,thefourth-generationdendrigraftpoly-L-lysinepolymer and the seven-membered homopolymer of asparticacidand5(6)-carboxyfluoresceinbioconjugatewerenecessaryto work in presence of complex biological fluids in order toensure sufficient binding between the partners. Since thefollowing experiments were all performed in a much lesscompetitivemedium(10mMHEPESbuffer,pH7.8),themoreaccessibleDGLG3andshorterD3CFwerechoseninthisstudyin order to keep these assays forGAGs as simple as possible(Fig.1).7Weenvisionedthat,usinganarraythatwasbasedonthe variable loading of the fluorescent indicator on thereceptor, each GAG could generate a specific fluorescentresponse pattern. Indeed, because they display specificelectrostatic surfaces, the complexes can be considered as“differential”receptorsforGAGs(Fig.2).8,9,10

Fig. 2 Minimized structures of G3-D3CF complexes at various loadings, withelectrostaticpotentialsurfaces.Loadings:100%(A),75%(B),50%(C),25%(D).9Forcalculationdetails,seeESI.†

UponadditionofdextransulfatetoG3-D3CFcomplexesinwater containing 10 mM HEPES and buffered to pH 7.8,distinct optical behaviours were detected depending on theloading of the indicator on the receptor (Fig. 3).11 At 100%loading, increased levels of dextran sulfate correlatedpositively with the fluorescence intensity (FI) response (Fig.3A). At 75% loading, a decrease of the FI occurred uponaddition of dextran sulfate until a minimum value FImin (Fig.3B), followed by the restoration of the optical signal. Similartitrationcurvescouldbeobservedatlowerloadings,althoughreaching FImin required increasing amounts of dextran sulfate(Fig.3Cand3D).Forallthesetitrationcurves,itcouldbenotedthat i) the initial fluorescence FI0 is in agreement with thetitrationcurveofG3withD3CF(seeESI†,Fig.S1),andii)afinalplateau with a specific maximum fluorescence value FImax isreachedataGAGconcentrationof4.8mg.L-1. A simple model to explain such unexpected behaviors,whichdependontheloadingoftheindicatoronthereceptor,isproposed(Fig.4).

Fig. 3 Fluorescence titrations of the G3-D3CF complexes with dextran sulfate.Loadings:100%(A),75%(B),50%(C),25%(D).9Dashed lines:grey=FI0,blue=FImin, red = FImax. Conditions: 10 mM HEPES buffer, pH = 7.8, [G3] = 163 nM([lysine]=20mM),λex=485nm,λem=535nm.

Fig. 4 Proposedmodel for thevariousbindingbehavioursofGAGs toG3-D3CFcomplexes.

Startingfromafully loadedG3withD3CF,FI0 isequaltoFIminsince the distance between the indicators on the receptor’ssurface is at its smallest, resulting in maximum fluorescenceself-quenching(Fig.4,top).Thesubsequentstepwiseadditionof GAG leads to the continuous displacement of the indictorfromthedendrigraft’ssurfaceandtotheincreaseofFI,untilaplateau that indicates thatall the fluorophore isactually freeinsolutionaccordingtothecalibrationcurveofD3CF(seeESI†,Fig. S1). In contrast, a lower loading gives a less compactedstarting arrangement of the indicators on the dendrigraft’ssurface, yielding a higher FI0 (Fig. 4, bottom).AdditionalGAGmolecules can therefore bind to the receptor withoutdisplacing but with compacting the fluorophores on thereceptor’s surface. This results into the diminution of theoptical signal until FImin that is correlated with maximumcompaction. Upon this point, additional GAG molecules

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restitute the indicator-displacement regime. In this model, i)the lower the loading is, the higher the GAG concentrationnecessarytoreachfullcompactionis,andii)thesameamountofGAG isneededto fullydisplacethe indicatorwhatever theloadingis. Suchamodelcouldbeusedtovirtuallyexplainthesimilarbehaviors that were observed upon the addition of otherGAGs, each one displaying specific indicator-displacement orindicator-compaction regimes (see ESI†, Fig. S2). As aconsequence, differential optical responses were observedwhentheselectedGAGsweresubmittedataconcentrationof3.5mg.L-1tooursensorarray(Fig.5).

Fig.5FluorescenceintensitiesfromtheadditionofGAGstoG3-D3CFcomplexes.Loadings:100%(A),75%(B),50%(C),25%(D).Conditions:10mMHEPESbuffer,pH=7.8,[G3]=163nM([lysine]=20mM),[GAG]=3.5mg.mL-1,λex=485nm,λem=535nm.

Differentiation was demonstrated and validated by lineardiscriminant analysis (LDA), which is a supervised procedurethatallows theclassificationofdata.12 Ina firstplace,knownsamples of GAGs (Fig. 6, triangles) were used as a traininginput to the procedure, giving a captured variance ratio of93.4%forthefirstlineardiscriminantand6.1%onthesecondone. Decision boundaries (Fig. 6, dashed lines) were thengenerated by using Bayes’ rule.13 These boundaries delimitareasofthereducedspacethatareexclusivelyattributabletoa single GAG. Finally, unknown samples (Fig. 6, circles) weretestedagainstthetrainingset,achieving100%ofidentificationwith50correctcasesoutof50,anddemonstratingthereforetherobustnessofthisassay.14Inordertofurtherdemonstratethe applicability of our sensor array, we tested its ability toassess the purity of samples of heparin contaminated with10%,20%,30%,and50%ofchondroitinsulfateB,whichisthemost prevalent impurity in pharmaceutical heparinpreparations.15Fromtheresultingscoreplot itwasclear thatallmixtureswereseparatedwithgoodresolution(Fig.7). In conclusion, we reported a simple sensor array thatallowsnotonly theunambiguous identificationof structurallysimilarpureglycosaminoglycans,butalsothedifferentiationofmixtures of glycosaminoglycans. The simplicity of this arrayrelies on the use of only one commercial receptor and onehighly accessible fluorescent indicator. Furthermore, a newsensing approach is introduced in this study that one coulddescribe as a compaction/displacement-indicator assay. We

are confident that such an original approach will find futureapplicationsinthesupramolecularanalyticalfield. This work was supported by the Centre National de laRecherche Scientifique (CNRS) and the Université deMontpellier. DGL G3was supplied by the COLCOM company(Montpellier, France). The authors thank Dr Sophie Sisavath(pharmacist) and Prof. Jean-Noël Bacro (mathematician) forfruitfuldiscussions.

Fig.6 LDAcanonicalscoreplot for theresponseof thesensorarraytoGAGs.Dashedlines: decision boundaries, triangles: training set, circles: blind tests, ellipses:confidencelimitsat99%forthetrainingset.

Fig. 7 LDA canonical score plot for the response of the sensor array toheparin/chondroitinsulfateBmixtures.Ellipses:confidencelimitsat99%.

Notesandreferences1 N.Volpi,Curr.Med.Chem.,2006,13,1799.2 (a) S. Beni, J. F. K. Limtiaco and C. K. Larive,Anal. Bioanal.

Chem.,2011,399,527; (b)M.Guerrini,Z.Zhang,Z.Shriver,A.Naggi,S.Masuko,R.Langer,B.Casu,R.J.Linhardt,G.TorriandR.Sasisekharan,Proc.Natl.Acad.Sci.U.S.A.,2009,106,16956.

3 D.B.Blossom,A.J.Kallen,P.R.Patel,A.Elward,L.Robinson,G.Gao,R.Langer,K.M.Perkins,J.L. Jaeger,K.M.Kurkjian,M. Jones, S. F. Schillie, N. Shehab, D. Ketterer, G.Venkataraman,T.K.Kishimoto,Z.Shriver,A.W.McMahon,K.F.Austen,S.Kozlowski,A.Srinivasan,G.Turabelidze,C.VGould,M. J. Arduino and R. Sasisekharan,N. Engl. J.Med.,2008,359,2674.

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4 (a) M. Twomey, T. Vokatá, M. R. Kumar and J. H. Moon,Chem.Commun.,2015,51,4065;(b)S.G.Elci,D.F.Moyano,S. Rana,G. Y. Tonga, R. L. Phillips, U. H. F. Bunz andV.M.Rotello,Chem.Sci.,2013,4,2076; (c)E.K.Nyren-Erickson,M. K.Haldar, Y.Gu, S. Y.Qian,D. L. Friesner and S.Mallik,Anal.Chem.,2011,83,5989;(d)1R.B.C.Jagt,R.F.Gómez-BiagiandM.Nitz,Angew.Chemie-Int.Ed.,2009,48,1995.

5 J.-P. Francoia, R. Pascal and L. Vial,Chem. Commun., 2015,51,1953.

6 B.T.NguyenandE.V.Anslyn,Coord.Chem.Rev.2006,250,3118.

7 Given that the simple carboxyfluorescein was not able tobind efficiently G3 in HEPES buffer (10 mM, pH 7.8),5 weselected an intermediate peptidic chain length for theindicator. See ESI for the straightforward solid-phasesynthesisofD3CF.†

8 A.T.WrightandE.V.Anslyn,Chem.Soc.Rev.2006,35,14.9 LoadingswerecalculatedfromthetitrationcurveofDGLG3

withD3CF,seeFig.S1 inESI.†Fortherationalizationof thetitrationcurve’sprofile,seeref.5.

10 Upon addition ofG3 toD3CF, a red-shift of the absorptionspectrum of the dye was observed by UV-visiblespectroscopy(λmax=493→501nm),indicatingitsstabilizationbynoncovalentinteractions(i.e.,thereductionofitsHOMO-LUMO gap energy) and confirming the formation of acomplexbetweenG3andD3CF.

11 Mass concentrations for GAGs were used in this study inorderto: i)beabletoblindtestthepolysaccharides,and ii)getridofanyapproximationabouttheirstructurethatmayleadtoroughmolarconcentrationcalculations.

12 S.Stewart,M.A.IvyandE.V.Anslyn,Chem.Soc.Rev.2014,43,70.

13 T. Hastie, R. Tibshirani and J. Friedman, The Elements ofStatisticalLearningData,2ndEd.,Springer:NewYork,2009.

14 APythonscripthasbeenedited inordertoautomatizethiswholeanalyticalprocedure,whichisbasedontheLDAclassof the scikit-learn Python module (http://scikit-learn.org),see: F.Pedregosa,G.Varoquaux,A.Gramfort,V.Michel,B.Thirion, O. Grisel,M. Blondel, P. Prettenhofer, R.Weiss, V.Dubourg, J. Vanderplas, A. Passos, D. Cournapeau, M.Brucher,M. Perrot and É. Duchesnay, J.Mach. Learn. Res.,2011,12,2825.Thescriptisavailableuponsimplerequest.

15 G.ANeville,F.Mori,K.R.HolmeandA.S.Perlin,J.Pharm.Sci.,1989,78,101.

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