WERNER PRIZE 2007/GRAMMATICAKIS-NEUMANN PRIZE 2007/ANALYTICAL CHEMISTRY417 doi:10.2533/chimia.2007.417 CHIMIA 2007, 61,No. 7/8
Werner Prize 1 Gramaticakis –Neumann Prize 2007 2
From Simplicity to Complexity via Subcomponent Self-Assembly Photoinduced functions in multicomponent molecular systems
Jonathan Nitschke,Marie Hutin, David Schultz,RupamJ.Sarma, SerenaSilvi,Alberto Credi* Victoria E. Campbell, Gérald Bernardinelli Dipartimento di Chimica G. Ciamician, Università degli Studi di Bologna, Organic ChemistryDepartment, University of Geneva, 30 quai Ernest- via Selmi 2, I-40126 Bologna,Italy Ansermet, 1211 Genève 4 Light-induced processesare at the basis of fundamental natural phenomena Thecreation of complexstructures from simple building blocks via ther- as wellasofavariety of applications.Since thefunctions that can arise modynamic self-assembly requires an understanding of the rules followed from the interaction between light andmatterdependonthe degreeofcom- duringthe self-organizationprocess. Since theinceptionofthisresearch plexity and organizationofthe receiving matter, theresearch on these program in August2003, we have sought to identify theserules and to apply processeshas progressively moved frommoleculartosupramolecular(mul- themtosynthetic problems.Subcomponent self-assembly1 describessys- ticomponent) systems.Examples of multicomponent systems capable to tems in which simple amines and aldehydesmay be induced to come to- perform specific functions under light stimulation (photochemical molecular gether around metal-ion templates via the formation of imine (C=N) devices, PMDs)have been developed [1], relying on processessuch as pho- bonds. 2,3 We have thus been abletocreatecomplex structures such as a toisomerization and photoinduced electron or proton transfer. 4 catenane (below, right) or apair of helical molecules linked at aright angle Herewewill describeafew recent examples of PMDs studied in our labora- through metal coordination (below,left),having atertiarystructuresimilar tories[2],designed to (i)processbinaryinformation (molecular logic gates to that of aprotein. Thesestructures arecapable of dynamic reassembly at 5 andcircuits)or(ii) undergo controllablemotions of some molecular com- both N Metal and C=Nlinkages. ponentswith respect to the others(molecular machines). Apart from futuristic applications,investigations on PMDs can increasethe H H N basic understanding of avariety of processes, as well as develop reliable N theoretical models.Thisresearch hasalsothe important merit of stimulating O O theingenuity of chemists,thereby instilling newlifeinto chemical sciences. N N N Cu+ N N Cu+ N N N O O [1] V. Balzani, A. Credi, M. Venturi, Molecular Devices and Machines , Wiley-VCH, Weinheim, 2003.A.Credi, Aust.J.Chem. 2006, 59,157. N H H N [2]V.Balzani,M.Clemente-León, A. Credi,B.Ferrer, M. Venturi, A.H. 1. J.R. Nitschke Acc. Chem.Res. 2007,40, 103 Flood, J.F. Stoddart, Proc. Natl.Acad. Sci.USA 2006, 103,1178.R. 2. J.R. Nitschke,M.Hutin,G.Bernardinelli Angew. Chem.Int.Ed. 2004,43, 6724 Ballardini, A. Credi, M.T. Gandolfi, F. Marchioni, S. Silvi, M. Venturi, 3. M. Hutin, G. Bernardenelli,J.R.Nitschke Proc.Natl. Acad. Sci. USA 2006,103, 17655 Photochem. Photobiol.Sci. 2007, 6 ,345.S.Silvi,E.C.Constable, F.M. 4. M. Hutin, C.A. Schalley,G.Bernardinelli,J.R.Nitschke Chem.Eur.J. 2006,12, 4069 Raymo, A. Credi, submitted. 5. D. Schultz,J.R.Nitschke Proc.Natl.Acad. Sci. USA 2005,102, 11191
Analytical Chemistry 3 Analytical Chemistry 4
Snapshots of aheterogeneous catalystatwork: From integral Chemometrictoolstosimplifymethoddevelopment:screeningofdop- towardsspatially resolved spectroscopicmonitoringofsolid materials ing agentsinurinary samples.
Jan-DierkGrunwaldt1 ,Stefan Hannemann1 ,BertramKimmerle1 , Ivano Marchi,Serge Rudaz, Jean-LucVeuthey Alfons Baiker1 ,Pit Boye2 ,JensPatommel 2 ,Christian G. Schroer2 LaboratoryofAnalytical Pharmaceutical Chemistry, School of Pharmaceu- 1 Institute forChemical andBioengineering, Department of Chemistry and tical sciences,University of Geneva, University of Lausanne, Bd d'Yvoy 20, Applied Biosciences,ETH Zurich, Hönggerberg, HCI, CH-8093 Zürich 1211 Geneva 4, Switzerland 2 Institute of Structural Physics,TUDresden, D-01062 Dresden Analysesinanti-doping control occur in two steps. First,ageneric To gain information on the structureofsolidmaterials such as heterogene- fast screening analysis is used to determinethe presence of alarge number ouscatalysts,insitu X-rayabsorption spectroscopy (XAS)isawell-suited of forbidden compounds in urine. Second,inthe caseofpositive result, a technique. In certain processesthe structureofacatalystmay varyalong a specific procedure(confirmatoryanalysis)has to be performed to quantify catalytic reactor particularly if prominent gradients in temperatureorcon- thesubstance(s). centration occur.The investigation of these phenomena requires spatially Screening method development is tedious and time consuming due resolved techniques on amicroscale. to the necessity to optimizethe sample preparation of alarge quantity of Here we present structural data of noblemetal catalysts during partial oxida- compounds.The useofchemometric toolsisthereforeproposed to reduce tionofmethane (CPO). To efficiently recordXAS spectra in alocally re- the number of tested analytes in method development by choosing represen- solved way, anew approach is suggestedusing aCCD-camera[1].The re- tative compounds of the whole set. sults arecomparedtothosefrom XAS-measurements. In addition,the In this study, agroup of thirty-sixdoping agents consisting of diuretics temperaturegradientwas determined usinganinfraredcamera[2].Acor- and beta-blockerswas used. Astandardsolution containing all analytes was relation between the structure of the catalyst, the catalytic data andthe axial loaded, washed and eluted with four different SPEsorbents.All fractions temperatureprofile in the catalytic reactor was established. Tremendous were collected and each compound wasretrieved by LC-ESI-MS. In order structural changes of the noble metal particles within agradient of lessthan to bringout differences among compounds,amultivariate analysis approach 100 mm thicknessand astrong dependence of thegradient on the reaction was used. Asmall number of groups emerged and subsequent method de- conditions (temperature, space velocity)wereobserved. Veryrecently, velopment was performed by selecting representative compounds in the "snapshots" werenot only taken of the variation in catalyststructureand obtained clusterisation. temperature, but we even succeeded in following thesechanges in a time-resolvedmannerusing aCCD-and an IR-camera.
[1]J.-D. Grunwaldt, S. Hannemann, C.G. Schroer, A. Baiker, J. Phys. Chem. Lett. B 2006, 110, 8674; J.-D.Grunwaldt, S. Hannemann, A. Baiker, P. Boye,C.G. Schroer,Highlights of Analytical Chemistry, Chimia 2006, 60, 709. [2]S.Hannemann, B. Kimmerle, J.-D.Grunwaldt, A. Baiker, P. Boye,C.G. Schroer,inpreparation. ANALYTICAL CHEMISTRY418 CHIMIA 2007, 61,No. 7/8
Analytical Chemistry 5 Analytical Chemistry 6
Microchip electrophoresis bioanalytical applications Development of amethodtocharacterize nanoparticle uptakeinto Corn plants Marketa Vlckova, MariaA.Schwarz Karin Birbaum 1 ,RobertBrogioli1 ,Ludwig Limbach2 ,EnricoMartinoia3 , University of Basel, Spitalstrasse 51, 4056 Basel, Switzerland Wendelin Stark 2 ,Detlef Günther1
Theimplementation of microfabricated separation devices (socalledmicro- 1 LaboratoryofInorganic Chemistry, ETH Zurich,Wolfgang-Pauli Strasse chips)inelectrophoresis has become atrend in modernseparation science. 10, 8093 Zurich,Switzerland However,the miniaturization of the separation to microchip brings along 2 Institute for Chemical andBioengineering,ETH Zurich, Wolfgang-Pauli some benefits (speed,negligible consumption of substances or high Strasse 10, 8093 Zurich,Switzerland throughput analysis)alsosome drawbacks(limited separation efficiency and 3 Laboratoryfor Molecular Plant Physiology, University of Zurich,Zolliker- selectivity, higherdetection limits or the absenceofautomation). strasse 107, 8008 Zurich, Switzerland Nevertheless, in some applicationareas theadvantages of themicrochip separations overcome the limitations.Adevelopmentofaselective andsen- Nanoparticles (NP) areincreasingly used in various applications e.g. as sitive detection, often involving expensive reagents,orafast development catalysts or millingagents.Thereforerisks of exposureduring production of aseparation method could be listed as the examples of such applications. processesand distribution in the environment requireclarification about the Specificmeasurementsfor particular purposes (affinity measurementsand potentialrisks.Studies using human lung cellshaveshown that agglomera- other)represent then further useful applications of microchips tion, sedimentation and diffusion of NP affect uptake by cells [1]. This Thelisted applications areas of microchips willbedemonstrated by present- study has investigated the uptake of CeO 2 NP during exposuretoairborne ing the results of our research.The sensitivity of amperometric detection of NP into Corn plants. catecholamines has been specifically enhanced with thehelpofenzymes [1] Forthe experiments,the plants wereplaced in aglove box that allowed the andalsocarbon nanotubes [2]. Acomplex separation method has been de- production of NP flame spraysynthesis in situ forashortduration (~1min). veloped forsimultaneous separation of allthree catecholamines and their TheNPare distributed within the glove box by afan.The plants remain cationic metabolites [2]. Theseparation method together with the developed insidethe glove box foracertain time beforedetermining the amount of sensitive detection wasthensuccessfully appliedfor the measurement of the CeO2 that was taken up by the plant cells. Due to the low amounts that are level of thesecompounds in the mousebrain sample [2]. Aspecific applica- taken up by the plants,inductively coupled plasma massspectrometry(ICP- tion of the microchips will be represented by isoelectric focusing measure- MS)isusedtodetermine Ce-concentrations in theplant material. Sample ments on amicrochip,which areasubject of our ongoing research. Using a preparation includesawashingstepinorder to discriminate between NP whole-column imagedoptical detection with the microchip, acrediblede- thatwereenter thecells form surface contaminants.Details of thewashing termination of the isoelectric points of selected proteins by isoelectricfocus- procedureand results optained willbediscussedinthis presentation. ing wasfeasible.
[1]Vlckova M.,SchwarzM.A., Electrophoresis 2005, 26,2701. [1] Limbach L., Environ. Sci. Technol. 2005, 39,9370. [2]Vlckova M.,SchwarzM.A., J. Chromatogr.A 2007, 1142,217.
Analytical Chemistry 7 Analytical Chemistry 8
TandemMassSpectrometryDataMining: ProductIon Abundances Imaging and chemical analysis of biological nanostructures usinga Provide Complementary Peptide and Protein Structural Information setupfor tip-enhanced Raman spectroscopy(TERS)
YuryO.Tsybin and Hisham Ben Hamidane Thomas Schmid,Boon-Siang Yeo,Weihua Zhang, Renato Zenobi
Biomolecular Mass Spectrometry Laboratory, EcolePolytechnique Federale Department of Chemistryand Applied Biosciences,ETH Zurich, de Lausanne, FSB ISIC LSMB BCH 4307, 1015,Lausanne, Switzerland 8093 Zurich,Switzerland
Revealing the information hidden in the production abundance distribution Biological samples can be highly heterogeneousonthe nanometer scale. in tandemmassspectrometry in generaland in electron capture/transferdis- Bacterialadhesion andbiofilm formation, forexample,are controlledby sociation (ECD/ETD)inparticularcould providesubstantial benefits for nanometer-sized structures,suchaspili, flagella, and extracellular polym- peptide and protein structural analysis andimprove fundamental under- eric substances(EPS).The arrangement of thedifferent EPS(e.g. polysac- standing of gas-phaseradical ion chemistry duringECD/ETD.Herewere- charides, proteins,humic substances)inthe biofilm matrix is largely un- portonarecent progressinthis direction based on anovel insight into cor- known, becausemicroscopy techniques used so farwererestricted in spatial relation of ECD/ETD radical and prime product ion yield with solution- resolution to the micrometerand highernanometer range (e.g.confocal la- phasepeptide and protein secondarystructure. ser-scanning microscopy, CLSM)ordid not provide any chemical informa- First,comparison of radical/prime product ion abundance ratio in tion (e.g.atomic forcemicroscopy, AFM). Abetterinsight into the distribu- ECDversusactivatedion (AI)-ECD demonstratesastrong andreproducible tion of the different extracellularnanostructuresisimportant for the im- dependence on ion internalenergy. This dependence allows forproduct ion provement of biocidesaswellasfor processoptimization in wastewater type (N-terminalorC-terminal) determination in many cases greatly facili- treatment. tating de novo peptide sequencing and protein identification. AcombinedCLSM-AFMsetup allows AFMimaging of selected areas of a Second, targetedanalysis of ECD/ETD product ionabundances of CLSmicrograph with nanometer-scaleresolution revealing bacteria,pili, several peptides revealsaqualitative correlation between product ion yield flagella,and EPS. For nanometer-scalechemical analysis,the feasibility of and amino acid hydrophobicity. For linear peptidesitopens the way toward tip-enhanced Raman spectroscopy (TERS) was demonstrated, whereAg- sequence-based prediction of ECDfragmentationpatterns. Current status of coated AFMtipsinthe laserfocus enhance theRaman signal,yielding method validation on selected peptide librarieswillbepresented. chemical information with alateral resolution of down to 2050 nm. Algi- Furthermore,ECD product ion distribution correlates with suggested nates in the form of nanometer-sized fibersand hydrogels wereusedas solution-phasesecondarystructure of several peptides and protein enzy- modelsamplesfor biofilms andinvestigatedsuccessfully by AFMand matic fragments, particularly foralpha-helical structures.Mechanistic stud- TERS. Other biochemical compounds under study areproteins and lipids. ies in this area aredirected towardimproving our understanding of the ECD Thegoal of further studies is to improve TERStobecome arobusttool for processand progressing towardpossible site-specific secondarystructure theanalysis of biological samples, which allows,for example, theelucida- inferredfromECD data. tion of the distribution of different biopolymers(e.g. polysaccharides and proteins)inside the biofilm matrix andcan be applied in many other fields of biology and medicine. ANALYTICAL CHEMISTRY419 CHIMIA 2007, 61,No. 7/8
Analytical Chemistry 9 Analytical Chemistry 10
Lipophilicity indexmeasurementofpolar compounds using hydrophilic Auto-oxidation of vivianiteand amorphous Fe2+ compounds interactionliquidchromatography in dried sewage sludge granules
Bruno Bard, Sophie Martel, Pierre-Alain Carrupt Martine S. Poffet,BernardGrobety a ,Kurt K¨aserb ,Titus A. Jenny Department of Chemistry,UniversityofFribourg Unité de Pharmacochimie, Sectiondes sciences pharmaceutiques, a Department of Geosciences Université de Genève,Université de Lausanne, Quai Ernest-Ansermet 30, b Department of Industrial Technologies, UniversityofApplied Sciences CH-1211 Genève 4, Suisse. CH-1700 Fribourg, Switzerland Lipophilicity is one of the mostusedphysico-chemical parameterstobuild structureactivity relationships (SAR). Methods to measure, with agood Amain portionofdriedsewage sludge is incinerated in cementindus- confidence level, the lipophilicity of verypolar compounds arestill missing tries. This importantwasteisreduced to ashes and its energetic content except theobsoletethinlayer chromatography (TLC)[1]. is used as an auxiliaryfuel. During its storage, asignificanttemperature In this study, the retention factors(logk)of26neutral polar compounds increase occurs whichinduces sometimes athermal runaway. weremeasured using hydrophilic interaction liquidchromatography (Hilic), Responsible for this sudden temperature increase is acascade of chem- and wereevaluated as lipophilicity indices to measurelog P oct .Alinear sol- 2+ vation energy relationships (LSERs)analysis was performed to elucidate the ical events whichisinitiated by the oxidation of Fe ,representing an intermolecularforces controlling the retention in Hilic. importantpart of total iron (Fe tot ≤ 10 %driedmass ratio). As shown by XRD,alarge fraction of this ferric iron consists of microcrystalline
vivianite (Fe 3 (PO4 ) 2 · 8H2 O) [1]. We hypothesize thatthe heat release Therelativecontributionofeach parameter,obtained by standardization, 3+ shows thatsignificantdifferences liesbetween partitioning in n- of this oxidation combined withthe catalytic effect of the resulting Fe octanol/water system [2]and Hilic interactions.The H-bond donoracidity triggers the auto-oxidation of the organic matter (50 %solid content). parameter( ), is particularlymoreimportant in Hilic. Driedsludge granules areexcellentthermal insulatorswith lowthermal Theresults show thatlog kinHilic is not aparameter of choicetomeasure heat capacities.This explains the occurrence of amajor temperature in- the log P of polar compounds. However,theseretention factorsprovide oct crease within big storage tankseveninthe case of asmallenergy release. interesting information which could be used as alipophilicity/polarity pa- rameterinSAR. This work studies therefore the evolution of the Fe2+/Fe 3+ ratioduring storage of the dried sludge granules using ion liquid chromatography [2] [1]S.Gocan,G.Cimpan,J.Comer,in 'Advances in Chromatography',Eds. and the decomposition of vivianiteusing X-raydiffraction. The kinetics E. Grushka and N. Grinberg, Taylor &Francis Group, Boca Raton, of this Fe 2+ oxidation will be importanttounderstand the mechanism 2006,79-176. of spontaneous temperature increase. [2]C.Stella, A. Galland,X.Liu,B.Testa,S.Rudaz, J. L. Veuthey, P. A. Carrupt,J.Sep.Sci., 2005,28, 2350. [1]E.Frossard, J. P. Bauer,F.Lothe, Wat. Res., 1997, 31,2449. [2] X. Meng et al. , Environ. Sci. Technol., 2001, 35,3476.
Analytical Chemistry 11 Analytical Chemistry 12
Studyofnon-covalent complexes between human kinases and clinical Fast log Pmeasurementbyultra performanceliquid chromatography inhibitors by nanoelectrospray mass spectrometry (UPLC)
Jecklin M.C. 1 ,Touboul D. 1 ,JainR. 2 ,TallaricoJ. 2 ,and Zenobi R. 1 Yveline Henchoz a),b) ,DavyGuillarmea),Flavia Badouda), Jean-LucVeutheya),Pierre-Alain Carruptb) 1 Department of Chemistryand Applied Biosciences,ETH Zürich, CH-8093 Zürich, Switzerland. a) b) 2 LaboratoiredeChimie Analytique Pharmaceutique, Unité de pharma- Novartis Institutesfor Biomedical Research,GlobalDiscovery Chemistry, cochimie, Section des sciences pharmaceutiques,Université de Genève, Lead Synthesis &Chemogenetics,Cambridge MA,USA. Université de Lausanne, CH-1211 Genève 4, Suisse, Proteinkinases play akey role in cell regulation mechanisms and abnormal Asuitable pharmacokinetic profile,particularlyinterms of ADMET phosphorylationisoften acauseorconsequence of disease, like cancer and properties is required to obtain valuabledrugcandidates.Reversed-phase inflammation. Hence, protein kinases have becomeone of the mostimpor- liquid chromatography (RPLC) methods represent agood alternative to the tant drug targets in biomedical research. Our work is to develop amass spectrometrybased method to screen human kinase inhibitorsand classify traditionalshake-flask method to measure lipophilicity, akey parameter thembyaffinity. needed to definethese profiles[1].Moreover,LChas recently evolved with Experimental parameterswereoptimised in order to observe non-covalent the development of shortcolumns packed with smallparticles(sub-2 μ m) complexesinthe gasphase between LCKorp38 and inhibitors. For com- working at ultrahigh pressures(>400 bar), thus enabling fast and ultra-fast petitive experiments [1] with p38,the protein wasincubatedwith Bay43- analysis while maintaining agood efficiency in highflow-rate conditions. 9006, as reference compound. Twoother inhibitors(BIRB796, VX-745) In this work, an UltraPerformance Liquid Chromatography (UPLC) wereadded subsequently at thesameconcentrationasthe reference com- system able to workupto1000 bar was used forlog Pmeasurements.Dif- pound. When only the complex with the tighter binding ligand is observed, ferent newly derived stationaryphasespacked with 1.7 μ mparticleswere the concentration of the otherligand is adjusted. Arelative dissociation con- precisely characterized in termsofintermolecular forces (solvato-chromic stant can be calculated fromthe ratio of thepeakareas of thecomplexes. approaches)using awell-balanced setofcompounds.Isocratic as well as We obtained arelativeclassificationfor affinity in thegas phase gradient modes were tested, and theiradvantages and drawbacks pointed (BIRB796>VX-745>Bay 43-9006),which is in agood agreement with data out. TheUPLCapproach offeredasignificant increaseinthe throughput for in solution [2]. Thesameexperimentswereachieved with LCK, PD173074, the determination of lipophilicity parametersand thetestedcolumns exhib- as reference compound, and Glivec, Bay43-9006, Tarceva, Iressa,ascom- itedavery broadstability overanextendedpHrange (1
Analytical Chemistry 13 Analytical Chemistry 14
Lipidomic andmetabolomic study by mass spectrometryin UPLC-Q-TOF-MS/MSand CapNMR characterisationofbiomarkers correlationwithaging in C. elegans revealedbyametabolomic study of the woundresponsein Arabidopsis thaliana Johann Rivière ,María Eugenia Soria Díaz, Emmanuel Varesio and Gérard Hopfgartner G. Glauser1,2 ,E.Grata1,2 ,J.Boccard 3 ,D.Guillarme2 ,E.Marsden-Edwards4 , S. Rudaz2 and J.L. Wolfender1 Life sciencemassspectrometry, EPGL,University of Lausanne, University of Geneva, 1 LPP, 2 LCAP, 3 LCT, School of Pharmaceutical Sciences,University of Ge- Bd dYvoy 20, 1211 Geneva-4, Switzerland neva-Lausanne, Geneva, Switzerland, 4 Waterscorporation, UK
Theprocessofaging and longevity still remains achallenging and very Ametabolomic approach using LC-MSand capillaryNMR (CapNMR) has been devised to search fororiginal compounds involved in the defence complex biological issuewherethe nematode C. elegans has becomean mechanisms of the model plant Arabidopsis thaliana (Brassicaceae).The de important model system. It has been proved thecentral role of the novostructural determination of thesecompounds,mainly oxylipins,isvery insulin/insulin-like growth factor (IGF-1) signaling pathway in the challenging becausethey arepresent in minute amounts within the major regulation of its lifespan as well as in the flies,yeastand possibly in constitutive metabolites. Thus,anLC-MS-triggeredmicrofractionation pro- mammals where the vitellogenin genes vit-2 and vit-5 were identifiedas cedurefollowed by CapNMRwas used fortheir specific isolation and sub- daf-2/daf-16-regulated genes.Inrecent studies,apossiblelinkbetween sequent characterisation at the microgramlevel.Specimens of A. thaliana werewounded with forceps to mimic herbivoresattack and harvested after longevity, lipid metabolismand reproduction hasalsobeen investigated. 90 minutes. To identifydefence induced compounds,comparisons of ex- Preliminaryresults in our group based on differential proteomic studies tracts of control and wounded specimenswereassessedbyrapid UPLC-ES- using wild type N2 strain and Daf-16 C. elegans mutant showed up- TOF-MSanalyses,followedbymultivariateanalysis. Structuralinformation regulation of members of the vitellogenin proteins family. Consequently, about the putative biomarkerswas obtained usingUPLC-Q-TOF-MS/MS we evaluatedand developeddifferent analytical strategiesfor profiling analyses.However,isolationand characterisationbyNMR was necessary to confirmthe molecular structureofsome molecules.Because of the convo- metabolitesinthe two strainsbychip-basedinfusion approaches and lutednature of the extracts, an MS-triggeredmultiple step isolation proce- high performance liquid chromatography coupled to massspectrometry. durewas performed to avoid co-elutions of targeted compounds.Firstly, First, lipids from C. elegans were extracted by amodified Bligh-Dyer enriched extractswereseparated by semi-preparativeHPLCusing agradient method. In asecond time, identification andquantification of which was geometrically transferredfromthe UPLC gradient. To obtain phospholipids and sphingolipids of crude lipid extracts werecarry out by maximalresolution, selected fractions were further purifiedontwo semi- multiple precursorion scan (MPIS) in shotgun experiment using ahybrid preparative columns coupled in series under optimal isocratic conditions. Thelatterwerecalculatedfrom two precise gradients using HPLCmodel- quadrupole time-of-flight (Qstar)and atriple quadrupole-linear ion trap ling software. Thefractions containing wound induced compounds were mass spectrometers (Qtrap) with ananoflow ion source. Finally, dried, directly dilutedin5μ lofdeuterated solvent beforeanalysis by phospholipids,glycerolipids,sphingolipids,fatty acyls and daf-2/daf-16 - CapNMR. Extensive2DNMR experiments wereobtained from50-100 μ g regulated lipidic metabolites of each strain wereseparated by LC-MS. of the purecompounds.Inthisway, different polar oxylipins wereeffi- Data obtained were interpretedbyanalytical softwarestodetermine lipid ciently isolated and characterized. Thegeneric method developed can be andmetabolitesprofileswho highlightedsignificantchangesbetween appliedtothe investigation of anyminor bioactive constituentsinplants. wild type strain and Daf-16 mutant in C. elegans
Analytical Chemistry 15 Analytical Chemistry 16
Asymmetrical flowfield flowfractionationmultidetectionsystemasa Dynamic PreparationofSI-traceable CalibrationGas Mixtures tool fornatural biopolymer characterization Hans-Peter Haerri andManuela Quintilii Enrica Alasonati and VeraI.Slaveykova SwissFederal Office of Metrology (METAS) Environmental Biophysical Chemistry-ISTE -ENAC,Ecole Polytechnique Lindenweg 50, 3003 Bern-Wabern, Switzerland FédéraledeLausanne, Station 2, CH-1015 Lausanne, Switzerland Thedynamic preparation of standardgas mixtures by means of permeation Natural biopolymersare extremely difficult to analyzeand testbecausethey devices and dilution units is an accuratemethod forreactive substances like areusually presentascomplex mixtures with distributions in physicochemi- nitrogen dioxide or ammonia at very low concentrations as required fore.g. cal properties.The information obtained using non-separation based ana- airquality monitoring [1],[2].Microgravimetric characterisation of com- lytical tools is limitedsincemeasurementsare made on the entire mixture mercially available permeation devices,calibration of mass flows,purity and average values areobtained. Consequently, theproperty -performance analysis of the permeatesand of the complementarygases arefundamental relationships arealsolimited becauseitisnot clearwhether one or multiple for determining the combineduncertaintiesofthe calibration gasmixtures. components of thesemixtures contributetothe macromolecule properties. Themicrogravimetricapparatus,the traceability scheme forthe amount of Thepresent study explores the capabilities of theasymmetrical flow field substance fraction and the massspectrometricmethod arepresented [3], [4]. flow fractionation (aFlFFF) with amultidetectionsystemtocharacterize Results areshown from the studiesofthe conditioning times, the tempera- natural biopolymers. aFlFFF, coupled on-line to adifferential refractive ture dependenceand of the repeatability of permeation devices,the trace index and seven angle laserlight scattering (LS) detectorswas used to pro- analysis forthe purity of aNH 3 permeator and of various complementary vide information on the average values and continuous distributions of mo- gases [5]. An uncertainty budget with the individual sources is represented. lar masses, radius of gyration and hydrodynamic radius of severalnatural biopolymersincluding alginatesand succinoglycan. [1]ISO 6145-1to6145-11, Preparation of calibrationgas mixtures using Thehyphenation of an aFlFFF channeland aLSallows to obtain the mo- dynamic volumetric methods. lecular parametersinasingle runwithout the need forcalibration.How- [2]Immissionsmessung von Luftfremdstoffen Messempfehlungen, ever,managing such complex hyphenatedsystemrequires extensive knowl- Immissions de polluants atmosphériques -Recommandations pour le edge of themechanisms underlying each analyticaltechnique. In the aim of mesurage,Federal Officefor theEnvironmentFOEN acorrect interpretation of the experimental results and extraction of mean- http://www.bafu.admin.ch/luft/00632/00634/index.html? ingful molecular characteristics, theinfluence of key operating parameters, [3]M.Quintilii, proceedings 10ème Congrès International de Métrologie, including thecrossflowrate, thecarrier composition and ionic strength, the St-Louis,France 2001. accumulationwallmembranecharge and thesampleloadonthe retention [4]D.W.Zickert, metINFO9/3,2002, and molecular characteristicswas carefully evaluated. http://www.metas.ch/de/metINFO/2002/metINFO2002_3.pdf . [5]H.-P. Haerri,D.SchwallerSI-traceableMassSpectrometric Analysis of Warmthanks areextended to the SwissNational Science Foundation Gas Mixtures, Chimia2006, 60,No. 7/8, 381. PP002-102640 forproviding funding directly related to this work. ANALYTICAL CHEMISTRY421 CHIMIA 2007, 61,No. 7/8
Analytical Chemistry 17 Analytical Chemistry 18
IdentificationofTLC markers andquantificationbyHPLC-MS of variousconstituentsinNonifruit andcommercial Noni-derived )3*4501)& 0644&- !*"/( *"0 !&/(;6"/ "/( !6#&35 7 *3"6-5 "/% products "0)0/(*6 OlivierPotterat ,Roger von Felten,PeturDalsgaard, Matthias Hamburger* "#03"50*3& %> -&$530$)*.*&);4*26& &5 /"-;5*26&! $0-& 0-;5&$)/*26& =%=3"-& %& "64"//&! 5"5*0/ ! ,@?@ "64"//&! 6*44& Institute of Pharmaceutical Biology, University of Basel, Klingelbergstr.50, &1"35.&/5 0' )&.*453; "/% /45*565&0'*0.&%*$"-$*&/$&4! 6%"/ /*, CH-4056 Basel, Switzerland 8&34*5;! A?? )"/()"*! )*/" Sincethe approval of Noni juice as anovel food by the EC in 2003, prod- ucts derived fromNoni fruit ( Morinda citrifolia,Rubiaceae) arebecoming 09"%";4! ."44 41&$530.&53; )"4 */$3&"4*/(-;#&$0.&0/& 0' 5)& #&45 increasingly popular as food supplements [1]. While the knowledge on con- .&5)0%4 '035)& "/"-;4*4 0' $0.1-&: 1305&*/ .*:563&47 "53*:,44*45&% ", stituents of Noni fruit has considerably increased over recent years, quanti- 4&3 &40315*0/ 0/*<"5*0/ "44 1&$530.&53; , )"4 /"563"--; tative data remain scarce andthe chemical composition of commercial '06/% "1-"$& 0' $)0*$& */ 1305&*/ 130'*-*/( @ 7 / ! 3&$&/5 "%8"/$&4 products distributed mainly via Internet is poorly established. In the present )"8& 4)09/ 5)& 1044*#*-*5;503&1-"$& 03("/*$ ."53*$&4 #; */03("/*$ 0/&4 study, HPLCand HPTLC profiles of commercial Noni juices and capsules #"4&%0/*A 03 * A A! 7 &3&!9&3&1035 5)& %&4*(/ 0' "*A ,#"4&% werecompared. 3-Methyl-1,3-butanediol was identifiedasatypical marker 1)050,3&"$5*8& ."53*: '03 1)050,&-&$530$)&.*$"- */%6$&% $;45&*/&5"((*/( in Noni juices.The presence of sorbic acid (E200) was detected in one Noni %63*/( ,7 $56"--;! 4&-&$5*8& 5"((*/( 0' $;45&*/&4 *4 "/ *.1035"/5 juicedeclared as additive free. 453"5&(; */ 1305&*/ 130'*-*/( "4 *5 "--094"#&55&3 1305&*/ *%&/5*'*$"5*0/ We developed and validated an HPLC-MS method forthe quantitative 5)306() $;45&*/& 0/,-*/& $06/5*/( %63*/( 7 analysis of characteristic Noni constituents, including iridoid glucosides, scopoletin, rutin, fatty acid glucosides and anthraquinones.The separation was performed on aC-18columnwith agradientofacetonitrile in water
containing 0.1% formic acid, and detection waswith ESI-MS in the nega-
*
A tive ion mode. Themethod was applied to the analysis of various commer-
, cial juices and capsules.Significant differences were observed between the
samples. Asperulosidicacid, deacetylasperulosidic acidand rutin werepre-
sent in all products analysed, but theirconcentrations differedgreatly be-
tween the products.The fatty acid glucosides noniosides Band Cwerepre- sent in capsulesand mostjuices.Scopoletinwas mainly found in juices.The @ 7 &#&340-% "/% 7 "//! ! !@7 mutagenic anthraquinone alizarin whichhas been reported fromroots and A 7 &*/! 77 63*"+ "/% 7 *6<%"+! ! !A7 leaves was not detected in theinvestigated samples. 77 )&/! 7 7 )&/! ! !@7 7 ";0/! 7 0644&-! 7 36%&/5! 7 *0/ "/% 77 *3"6-5! [1]O.Potterat, M. Hamburger, Planta Med . 2007, 73,191. ! !A7
Analytical Chemistry 19 Analytical Chemistry 20
Extractionand Quantitative Analysis of Non-derivatized Glucosinolates Single molecule Tip-enhanced Raman Spectroscopy in PlantExtractsaValidated PLE/LC-MSProtocol Weihua Zhang,Renato Zenobi* Tobias Mohn, Olivier Potterat,MatthiasHamburger* LaboratoryofOrganic Chemistry, ETH Zürich, CH-8093 Zürich, Switzer- Institute of Pharmaceutical Biology, University of Basel, land Klingelbergstrasse 50, CH-4056 Basel, Switzerland Recently, single molecule tip-enhanced resonanceRaman spectroscopy of Glucosinolates have attracted significant interest due to thechemopreven- dye molecules has been claimed basedonthe observation of temporal spec- tive propertiesofsomeoftheir transformation products [1]. troscopic fluctuations.[1,2] However, tip-enhanced Raman spectroscopy Numerous protocols forthe extraction and analysisofglucosinolates have (TERS) of moleculesnot showing anyresonanceRaman effect is still diffi- been publishedbut little effort hasbeen devotedtooptimizeand validate cult, due to the extremely smallcross section of normalRaman scattering. crucialextraction parameters andsample preparation steps. Typically, the In this work, a2-mercaptopyridine sub-monolayer, which does not absorb plant material is heat-pretreated and/or extracted at high temperaturetoinac- visible light, was studied. Temporal intensity fluctuations of different vibra- tivate myrosinase,followedbyenzymatic desulfatation and analyis by re- tional modes were observed. This suggeststhat thesensitivity of our TERS versedphaseHPLC. More recently,analysis of intact glucosinolates has setup hasreached the single molecule level withoutthe additional resonance been attempted using ion-pair chromatography [2]. However,peak shape Ramaneffect.Thanks to the high sensitivity of TERS,studiesonbiological andseparation usually werenot satisfactory. samplesbecome possible.Weinvestigateddifferent bio-molecules,includ- We carriedout asystematicoptimization andvalidation of aquantitative ing amino acids and nucleic acids depositedonAusubstrates.Spectra com- assayfor the direct analysis of non-derivatized glucosinolatesin Isatis tinc- parable to the resultsbysurface-enhanced Raman spectroscopy (SERS) toria leaves(woad, Brassicaceae).Various parameterssuchassolvent com- were obtained. It provides an unprecedented opportunity to identify,monitor position, particle size, temperature, and number of required extraction steps and analyze labelfreebio-systems with nanometer spatialresolutionatam- were optimized using pressurized liquid extraction (PLE).Weobserved bient conditions. thermal degradation of glucosinolates at temperatures above50°C, and loss of >60% within 15min at 100°C,but no enzymatic degradation in the leaf samplesatambient temperature. Excellent peak shape and resolutionwas obtained by reversed-phasechromatography on aPhenomenex Aqua col- umn using 10mM ammonium formate as ion pair reagent. Detection was carried out by ESIMS(negativeion mode). Analysisofcruciferous vegeta- bles and spices such as broccoli(Brassica oleracea L. var. italica ), garden cress(Lepidium sativum L.) and black mustard ( Sinapis nigra L.) demon- [1]Zhang,W.; Yeo, B.;Schmid, T.;Zenobi,R.J.Chem. Phys.C2007, 111, stratedthe generalapplicability of the method. 1733. [1]B.Halkier,J.Gershenzon, Annu. Rev. Plant Biol. 2006 , 57,303.[2]L. [2]Neacsu, C. C.;Dreyer,J.; Behr,N.; Raschke, M. B. Phys.Rev. B 2006, Song et al.Anal. Biochem. 2005, 347,234. 73. ANALYTICAL CHEMISTRY422 CHIMIA 2007, 61,No. 7/8
Analytical Chemistry 21 Analytical Chemistry 22
Epitope mapping on bovine prion protein using chemical cross-linking Cationicterpolymers composedof andmassspectrometry bis1,3(N,N,N-trimethylammonium)2propylmethacrylate dichloride, acryloyloxyethyltrimethylammonium chloride andacrylamide: Tatiana Pimenova1 ,AlexisNazabal1 ,Bernd Roschitzki2 ,Jan Seebacher3 , Analysis of composition andelectrochemical behavior Oliver Rinner1 and Renato Zenobi1 R. Losada ,C.Wandrey 1 ETH Zürich, ETH Hönggerberg,8093 Zürich,Switzerland 2 FunctionalGenomics Center,UZH/ETH Zürich,8057 Zürich,Switzerland Ecole Polytechnique FédéraledeLausanne, Institut de Bioingénierie, 3 Institute forSystemBiology, Seattle, Washington 98103-8909, USA LaboratoiredeMèdecine Régénérative et de Pharmacobiologie, Lausanne, Switzerland
Developmentofnew analytical strategies for determination of epitopes, the Charged macromolecules,polyelectrolytes (PEL) areusedfor abroad vari- preciseregion of aprotein involved in the interaction with an antibody is ety of applications.Frequently,copolymersoreventerpolymershave better crucial to optimize diagnostic tools.Inthe presentstudy we have evaluated application performance than homopolymers. In such casesthe product anew analytical approach forcharacterizing epitopes using the monoclonal analysis and product quality control become achallenge,inparticular,ifthe antibody 3E7and bovine prion protein bPrP(25-241) [1]. Acombination of chemical structureofthe monomer units forming the polymer chain is very chemical cross-linking and mass spectrometrywas applied[2].Cross- similarand the polymers have high molarmasses. linking reactions wereconducted with isotope-labeled, amine reactive cross- This contribution presentsaprocedure to analyzequantitatively the compo- linkers, disuccinimidyl suberate(DSS-d0/d12) and disuccinimidyl glutarate sition of terpolymerscomposed of bis1,3(N,N,N-trimethylammonium) (DSG-d0/d6).After cross-linking, the covalently bound immuno-complex 2propylmethacrylate dichloride(dipole M),acryloyloxyethyltrimethylam- was analyzed directly usingaMatrix-Assisted LaserDesorption/Ionization monium chloride (Q9), andacrylamide combining FTIR andpotentiometric (MALDI)massspectrometerequipped withanIon Conversion Dynode titration. Theprocedure is basedonprevious calibration with appropriate (ICD) high-massdetector.The sample containing ahigh proportionofthe copolymers. Comparing the initial monomerfeedcomposition and the specific cross-linked complex wasdigested by chymotrypsin and the pep- product composition allows conclusions concerningthe monomer reactivity. tides obtained wereanalyzed by nano-LC-ESI-FTICR mass-spectrometry. Counterion activity measurements reveal the influence of the solution vis- As aresult, acomplete fading of the peak intensities corresponding to the cosity on the ion mobility. Overall, FTIR present apractically useful simple peptides representing the epitope was observed.The aminoacids 150-160 method to analysethe composition of these terpolymersand to control the areinvolved in the binding to the antibody. Ourresults showed excellent polymer production. agreement with pepscan data. Theepitope of 3E7onbPrP(25-241)was de- termined, with only alow amount of sample (200 picomoles)needed. Acknowledgements: Theworkwas supportedbythe SwissNational Science Foundation, grant 200021-107737.The monomerswereprovided by TAMINCO. [1] S. B. Prusiner, Science, 1982, 216,136. [2]A.Sinz, Mass Spectrom. Rev., 2006, 25, 663.
Analytical Chemistry 23 Analytical Chemistry 24
Analysis of endogenous metabolitesofasingle yeastcelllysate CZE andMEEKCcoupled with APPI/MSfor pharmaceutical applications Andrea Amantonico,Joo Yeon Oh and Renato Zenobi Julie Schappler,Davy Guillarme, Josiane Prat,Serge Rudaz, Department of Chemistry andApplied Biosciences, ETHZurich Jean-LucVeuthey
Metabolomics is ausefultool forhigh throughputanalysis of the biochemi- cal phenotypeofacell.Conventional analytical methods provide datathat LaboratoryofAnalytical Pharmaceutical Chemistry areaveragedovermanycells,while even cells in monoclonal cultures dis- School of Pharmaceutical Sciences,University of Geneva, University of play strong variations on all levels.Thus asingle cell approach to me- Lausanne, Bd d'Yvoy 20 -1211 Geneva 4-Switzerland tabolomicsisessentialtocreateaccuratemodelsthatconsider thestochastic nature of many cellularevents. Theelectrospray ionization (ESI)source is currently the method of choice Single cell analysis is difficult due to the minute amounts of samples in each forCE-MS becauseofits sensitivity, versatility andeaseofimplementation. cell. We areaddressing this challenge by couplingamicrofluidiccellproc- Nevertheless, ESIpresentssomeinherentdrawbacks mainly relatedtolarge essing and sample preparation step to anovel matrix assisted laser desorp- signal suppression which can occur with complex matrices or non volatile tion/ionization massspectrometry(MALDI-MS)method. We demonstrate additives. Therefore,partialfilling techniqueshavetobeimplementedfor thatthe combination of piezoelectrically-drivennanoliterspotting with thin chiral, micellar(MEKC) or microemulsion(MEEKC) analyses to avoid layerpreparation meetsthe needsofsingle cellanalysis in terms of sample background noiseand source contamination. Theatmospheric pressure pho- consumption and limit of detection. Themethod is alsotestedtodetermine toionization (APPI) sourceisdescribedtobelesssensitive to matrix effect metabolite levels in yeastcells extracts. Thespotting of the sample was andtothe presenceofadditivesand broadensthe range of ionizable analytes conducted by depositing avariable numberof500pL drops.Spotting of the to non-polar compounds.This ionization source is similar to APCIwith the solution premixedwith matrix wascomparedwith the spotting onto athin corona needlereplaced by akrypton discharge lamp.Inthisconfiguration, layer of matrix. Both methods showed alimit of detection (LOD) in the at- the sample is vaporized prior to ionization and non-volatile salts can be tomole range,atleasttwo orders of magnitude below the one obtained with therefore eliminatedduring this step. classicpipette spotting. Even spots with asamplevolumeasminuteas In this study, numerous CE-APPI/MS parameterswereoptimized thanks to 500pL gave adetectable signals of metabolites reaching forsome of them a an experimental design strategy. Genericconditions adapted forpolar as LOD(90amol foradenosine 5-triphosphate)comparable with the content in well as non-polar compounds werefound and limits of detectionwerede- asingle yeastcell. Thesamemethods werethen applied to yeastcell extract. termined in CZE-APPI/MSfor apharmaceutical mixture and comparedto Finally the spectrum of the cell extract corresponding to asingle cell was conventional CZE-ESI/MS.Furthermore,possibilities to enhance selectivity obtainedbyspotting 500pLofdiluted yeastcell extract. by the direct coupling of MEEKCwith APPI/MSwerehighlighted forthe complexseparation of ionized and neutral compounds in positive and nega- tive APPI modes.Systemstability as well as method sensitivity and effi- ciency wereemphasized. Application to screeninganalysis of doping sub- stances (such as -blockers,steroids,diuretics )was finally implemented. ANALYTICAL CHEMISTRY423 CHIMIA 2007, 61,No. 7/8
Analytical Chemistry 25 Analytical Chemistry 26
CE-LIF analysis of pharmaceuticalcompounds in biologicalfluids: Nanoscale molecular analysis and chemical imaging via atmospheric evaluationofvarious derivatizationmodes pressure near fieldlaser ablation mass spectrometry
JulieSchappler,Serge Rudaz, Jean-LucVeuthey Thomas A. Schmitz, GerardoGamez, Renato Zenobi LaboratoryofAnalytical Pharmaceutical Chemistry School of Pharmaceutical Sciences,UniversityofGeneva, University of Department of Chemistryand Applied Biosciences,ETH Zurich, Lausanne,Bdd'Yvoy 20 -1211 Geneva4-Switzerland Wolfgang-Pauli-Str. 10, 8093 Zurich, Switzerland
Analysis of pharmaceutical drugs andmetabolites at lowconcentration We present an instrument that achieves chemical analysis of samplesatat- levels in biologicalmatricesrequiressensitive methods sincemanydrugs mospheric pressure with nanoscale spatialresolution by coupling mass spec- show ahighvolume of distribution. In ordertoreach detectionlimits in the trometrytolaser ablation via scanning near field optical microscopy ng/mL range,laser-inducedfluorescence(LIF) washyphenatedtoCEto (SNOM). No other method availabletoday offers this unique analytical ca- ensure sensitivity andselectivity.However,few compounds possess a pability, sinceclassical techniquesfor the characterization of materials on strong andnativefluorescence, aderivatizationprocedureistherefore the nanoscale, such as standardAFM,STM and electron microscopy, yield usuallymandatory. little or no chemical information at all, andmosttraditionalchemical analy- In this study, threederivatizationprocedures were assessed forCE-LIF sismethods arefar fromhaving the required spatialresolution. analysis of pharmaceutical compounds in plasma (suchasmyorelaxants, stimulants, -blockers):liquidphase pre-capillary derivatization,solid phase As opposed to aprevious SNOM-MSdesignbyKossakovski[1] which re- pre-capillary derivatizationand on-capillary derivatization. Various laser quired the sample to be in high vacuum, our setup allows the analysis to be sourceswereevaluated (442 nm gaslaser vs. 410 nm diode laser),aswellas performed at ambient conditions.Inprevious workbyour group [2], we derivatizationtags(naphtalene-2,3-dicarboxaldehyde vs. fluorescamine) and electrophoretic modes(CZE vs. MEKC). Limits of detectiondownto5 demonstratedthe feasibility of nanoscale-MS at atmospheric pressure,but ng/mL in plasma werereached whichenhancedsensitivity compared to analysis at thattime waslimitedinmassspectralperformance. We have conventionalCE-UV.Finally,anotherCE-LIF method, without fluorescent now developedacombination of an optimized iontrapand time-of-flight tagging thanks to a266 nm diode laser, wasdevelopedand compared to MS [3]which allows foraccumulationofthe sampledmaterialand which otherapproaches. allows for simultaneous detection of awide range of mass-to-charge ratios.
Acharacterization of the instrument performancewith different samples will be presented. Thenear field ablation craterswith theircorresponding mass spectrometry signatureswill be demonstrated.
[1]D.A.Kossakovski, et. al., Ultramicroscopy 1998, 71,111. [2]R.Stöckle, et al., Anal.Chem. 2001, 73,1399. [2]P.D.Setz, T. A. Schmitz,R.Zenobi., Rev. Sci. Instrum. 2006, 77, 024101.
Analytical Chemistry 27 Analytical Chemistry 28
TowardsRapid Nanoscale Chemical Analysis using Tip-Enhanced LaserAblation-ICP-MS:Quantificationoffemtosecond laserablation Raman Spectroscopy with Ag-Coated DielectricTips generatedaerosols using solution nebulization forcalibration
Boon-Siang Yeo,Thomas Schmid,Weihua Zhang, Renato Zenobi* M. Wälle,J.Koch and D. Günther
Department of Chemistryand Applied Biosciences,ETH Zurich, LaboratoryofInorganic Chemistry, ETHZurich 8093 Zurich,Switzerland 8093 Zurich,Switzerland
Sincethe introduction of tip-enhanced Raman spectroscopy (TERS) in Theuse of nebulized liquid standards to quantifyLA-aerosols in the ICP- 2000,intenseresearch effortshave been madetopushthe frontier of this MS has firstbeenproposed by Thompson et al [1]and recently OConnor et technique towards reproducible nanometer scale chemical analysis.The al [2] show the usability of this approach for the majority of the elements for ability to perform such measurementsisofgreat importance, e.g.,for the different matrices. understanding of catalytic processesand forthe fabrication and quality con- Since femtosecond laser systemshave shown thereability to produce aero- trol of molecular electronics. sols whichare stoichiometric representative for the sample [3], in this study aTi:Sapphirefs-lasersystemoperated at its 3 rd harmonic (265nm) was used In this work, theinfluence of dielectricsubstrates on theRaman scattering to ablate conducting (brass) and nonconducting (glass) materials.For cali- activitiesofAgoverlayers hasbeen investigated. Materials with low refrac- bration, liquid standards arenebulized and applied directly or via amem-
tiveindices such as SiO 2 ,SiOx and AlF3 have been found suitable as sup- brane-desolvatisation unit to the plasma. Thedesolvatisation step wasim- porting platforms forAgfilms at 488 nm illumination to give strong sur- plemented to reduce spectralinterferences caused by higher oxide building face-enhanced Raman scattering fordye molecules. This finding is then rates through the addition of water to the plasma.Duringablation ablank extended to TERS.Huge observed enhancements of 70-80 ,corresponding solution wasnebulized to ensure thatplasmaconditions remain as constant to net enhancementsof > 104 ,havebeen achievedfor brilliant cresyl blue as possible. To compensatefor the different massflows fromthe nebulizer test analyte using Ag-coatedtips made from or pre-coated with low refrac- and the laser ablation unit,Calcium and Copper wereusedasinternal stan- tive index materials.The yield of tips giving significant enhancement to the dardsfor glassand brasssamples respectively. Raman signals is found to be closeto100%.Thesefindings arecrucial steps Most of the determined elements show adeviationofinthe range of 1% to towards theuse of TERSasarobusttechnique forrapid chemical imaging 10% againstthe certifiedreference valuesregardlessifitconcerns main or with nanometerspatialresolution. trace elements. However,especially in theglass samplessomeelementslike Zinc and Selenium show deviationsupto50%.
[1]M.Thompson,S.Chenery, L. Brett, J. Anal.At. Spectrom., 1989, 4 , 11-16 [2]C.OConnor,B.L.Sharp, P. Evans,J.Anal. At.Spectrom., 2006, 21, [1]B.S.Yeo,T.Schmid,W.Zhang,R.Zenobi, Anal. Bioanal.Chem. 556-565 2007,387,2655. [3]J.Koch, D. Günther, Anal.Bioanal.Chem., 2007, 387,149-153 ANALYTICAL CHEMISTRY424 CHIMIA 2007, 61,No. 7/8
Analytical Chemistry 29 Analytical Chemistry 30
UPLC at high temperatureasahighly efficienttechnique forthe CharacterizationofMonoclonal Antibodies: SurfacePlasmonReso- separationofcomplex pharmaceutical mixtures. nanceorHigh-mass MALDI MassSpectrometry?
Davy GUILLARME ,Dao T.-T.NGUYEN, SergeRUDAZ,Jean-Luc BICH Claudia 1 ,NAZABAL Alexis1,2 ,WENZELRyan1,2 ,SCOTT Michael3 VEUTHEY and ZENOBIRenato1
1 Department of Chemistryand Applied Biosciences,ETH,CH-8093,Zu- LaboratoiredeChimieAnalytique Pharmaceutique, Université de Genève, rich Université de Lausanne, 20,Bdd'Yvoy -1211 Geneva 4-Switzerland 2 CovalX GmbH,Technopark1,CH-8005, Zurich 3 FunctionnalGenomic Center of Zurich, CH-8057,Zurich Pharmaceutical industryhas alwaysbeeninterestedbyhighly efficient ana- lytical techniques,tosolve verycomplex pharmaceutical cocktails or in drug impurity profiling (i.e.todeal with ahigh numberofimpurities).Addi- Thecharacteristics of antibody-antigen interaction such as the natureofthe tionally,with thedevelopment of genomics, proteomicsand more recently epitope, theability of the antigen to bind two antibodies simultaneously and metabolomics, thereisaneed foranalytical procedures abletoyield high resolution in acceptable analysis time. the kinetic and dissociation constants areusually determined using Surface Among the existing solutions to improve efficiency in liquid chromatogra- PlasmonResonance(SPR). This method requires that interacting partnersbe phy (LC),the useofUPLC(UltraPerformance Liquid Chromatography) in separated:one is immobilized while theother is flowing acrossthe surface. high temperaturemobile phaseconditions (HT-UPLC) represents an ade- Thedevelopment of anew protocol using cross-linking chemistryassociated quateapproach to improve theefficiency while maintaininganacceptable with high-mass mass spectrometry using matrix assisted laserdesoprtion analysis time.Indeed,this approach is compatible with long columns ionization (MALDI MS)allows characterizationofmonoclonalantibodies packed with sub-2 μ mparticles, becausethe mobile phaseviscosity de- directly in solution. Samplesare stabilized withcross-linking chemistry creases with temperature. prior the analysis with ahigh massdetector system (CovalX). This procedurewas investigated in isocratic as well as gradient mode. In We characterized thebinding between the monoclonal antibody 1E5and the order to obtain high performance in HT-UPLC, acompromisehas to be found between column length,workingmobile phaseflowrate, efficiency bovine prion protein (bPrP) (sequence of the binding site, multibindingstud- and generated back pressure.This work presentssomeadvantages and iesand kinetic measurements) comparinghigh-mass MALDImassspec- drawbacks of HT-UPLCfor high resolution separations. trometry andSPR.Multibinding experiments were performedwith asecond With this approach, it is possible to reach ca. 100000 plates in isocratic antibody againstbPrP, and epitope mappingwas achieved by acompetition mode, with asuitable analysis time (lessthan 1hour), using a450 mm col- assaywith competing peptides. umn length packed with 1.7 μ mat90°C and 1000bar.Finally,HT-UPLC was appliedfor theseparationofcomplex mixtures(35 pharmaceutical compounds and 35 doping agents)ingradient mode, forcolumn length ranging from30to450 mm. Some limitations havebeen established in both modes, depending on the used column length (i.e. impossibility to work at optimal flow rate, potential compounds degradationfor very long separa- tions)and some rules aregiven to select the appropriate column length.
Analytical Chemistry 31 Analytical Chemistry 32
Your 1D 13CNMR experiments lasttoo long? Getting to the Bottom of Chemical Cross-linking: Tryour new 2D long-range experimentwithhigh-resolution in thecar- ReactivitiesofDifferent Amino Acids bon dimension! StefanieMädler ,Renato Zenobi
Bruno Vitorge,Damien Jeannerat Department of Chemistryand Biosciences,ETH Zurich, 8093 Zurich, Swit- zerland University of Geneva, Department of Organic Chemistry, 30 Quai E. An- sermet, 1211-Geneva 4, Switzerland Chemical cross-linking, in combination with Matrix Assisted LaserDesorp- tion/Ionization (MALDI)massspectrometry,has emerged as apowerful Most chemists have no time or availability to applysomeofthe most pow- tool forthe identification of noncovalent inter-orintramolecular interac- erfulNMR technique even when they provide significant resolution en- tions in proteins.Despite the large number of applications,only afew stud- hancementorreduction in experimentaltime.[1] They need ready-to-used iesconcerning the reactivity andselectivity of cross-linkers toward certain experiments.Wehave developed such anew 2D long-range experiment amino acids have been reported [1 -3]. In order to understand the specificity providing the high resolution of 1D carbon spectra andthe high-sensitivity and reactivity of different cross-linkers, systematic studies werepeformed of proton detected 2D experiments. on smallpeptides (upto10aminoacids)with theminimumnumber of reac- In standard2Dlong-range experiments,the accuracyofthe carbon chemical tive groups.NHS estersdescribed usually as amine-reactive, but sometimes shifts is usually not muchbelow one ppm makingitimpossible to resolve showing reactivity towards hydroxyl containing amino acids,wereapplied closecarbons.Wedeveloped acomplementary2Dexperiment providing to lysine, tyrosineand/or serine containing peptides.MALDI-MSallows a 20-fold resolution improvement in the carbon dimension. In the resulting rapid identification of the numberofamino acidsmodifiedbythe cross- spectra, nearly degenerated signals can be resolved and the values of the linker.The so-called decoration of theamino acids was highly dependent on carbon chemical shifts can to be extendedtotwo digits after the period. The the reaction conditions.The hydroxyl containingamino acids serine and newcomplementary experiment requiresnearly thesameexperimentaltime tyrosineappeared much moredifficult to modifythan lysineunder compa- as the standardlong-range experiment. rable conditions.The formation of intermolecular links between two peptide moleculeswas observedonly in exceptionalcases.Thisdemonstratesthat Application to asmall amount of amixtureoftwo naturally occurring natu- chemical cross-linking of biological moleculesismainly observedfor spe- ralproducts is used as ademonstration. cific protein/peptide interactions.
[1] C.L. Swaim,J.B.Smith, D.L. Smith, JAmSoc Mass Spectrom 2004, 15, 736. [2]M.D.N.Leavell, P. Novak, C.R. Behrens,J.S.Schoeniger, G.H. Kruppa, [1]D.Jeannerat, J. Magn.Reson. 2007, 186,112. JAmSoc Mass Spectrom 2004, 15,1604. [3] B.T. Miller, T.J. Collins,M.E.Rogers,A.Kurosky Peptides 1997, 18, 1585. ANALYTICAL CHEMISTRY425 CHIMIA 2007, 61,No. 7/8
Analytical Chemistry 33 Analytical Chemistry 34
Hydroxylated MetabolitesofHexachlorocyclohexanes:Bacterial For- Rolesofdynamic metal speciation and membranepermeability in the mation andStereochemical Configurations metalfluxatpermeation liquid membranes
Vishakha Raina, Mandeep Dadhwal, Hans Rudolf Buser, Daniel ZeshiZhang and Jacques Buffle Rentsch,Hans-Peter E. Kohler Analytical and Biophysical Environmental Chemistry(CABE) SwissFederal Institute forEnvironmental Scienceand Technology Chimie Minéral, Analytique et Applique,Sciences II,University of Geneva (EAWAG),CH-8600 Dübendorf, Switzerland and SwissFederal Laborato- 30 quai E. Ansermet, CH-1211 Genève 4, Switzerland ries forMaterials Testing and Research (Empa),CH-8600 Dübendorf, Swit- zerland. In natural waters,trace metals arepresent in alarge number of different forms. In order to understand their bioavailabilityfor microoganisms,the study of the role of different metal species on themetal flux related to bi- Although theuse of hexachlorocyclohexane (HCH), one of the mostpopular ouptake is akey issue. Permeation Liquid Membrane(PLM) is apromising insecticides after the Second World War,has been discontinued in many techniquefor trace metal dynamicspeciation, becauseitisasimple tech- countries, problems remain from former productionand waste sites. nique,its sensitivity is high due to its preconcentration capability andthe Cl OH OH processesofmetal transportthrough PLMare similar to thoseoccurring in Cl Cl Cl Cl Cl Cl biological membranes.InPLM the metal ion is transported through alipo-
Cl Cl Cl Cl Cl Cl philic liquid membrane, by complexation with alipophilic ligand which Cl Cl OH servesascarrier. Theimportant parameters whichinfluencethe metalflux arethe diffusion layer and membranes thicknesses, the diffusion coefficients of the metalion andits complexes, the chemical association anddissocia- Cl OH OH tion rate constants of the metalcomplexesinsolution, the partition coeffi- Cl Cl Cl cientofmetal ion between the solution andthe membrane,and the nature
Cl Cl Cl Cl Cl Cl and concentrationofthe carrier.Ageneralmodelrelating these parameters Cl Cl OH to the steady-state flux of metal through the PLM, has been developed. It is applicable to complexeswith anydegreeoflability (from fully labile to in- We isolated and characterized hydroxylated metabolitesfromHCH incuba- ert). This modelhas been validatedexperimentally with complexeswith tion experiments with the common soil microorganism Sphingobium indi- varying degreeoflability,inparticular: Pb-NTA, Pb-TMDTA, Pb- cum B90A [1]. Theregio- and stereo-selectivity of the hydroxylation Diglycolate, Cu-Diglycolate and Cu-N-(2-Carboxyphenyl)glycine com- mechanismisdiscussed.Several of the metabolitesweredetected in plexes. It enablestopredict the role of the different typesofnaturalcom- groundwater from aformerHCH production site in Switzerland. plexesonthe bioavailability of metals for organisms anditservesasatheo- [1]V.Raina,A.Hauser, H.R. Buser,D.Rentsch,P.Sharma, R. Lal, C. retical basisfor PLMsensors whichebnabletodetermine the ecotoxicologi- Holliger, T. Poiger, M. D. Müller, H.-P. E. Kohler, Environ. Sci. Technol. cal rolesofmetalsinaquatic systems. 2007, in press.
Analytical Chemistry 35 Analytical Chemistry 36
Investigation of aerosol transport parameters on signal responsein Determination of gas-phasebasicityofpeptidesand proteinsatatmos- LA-ICP-MS phericpressure by ESSI-MS
RobertKovacs,Detlef Günther D. Touboul,M.C.Jecklin, R. Zenobi
Trace Element and MicroAnalysis,LaboratoryofInorganicChemistry Department of Chemistryand Applied Biosciences,ETH Zürich,CH-8093 ETH Zürich,Wolfgang-Pauli-Strasse 10, CH-8093 Zürich,Switzerland Zürich, Switzerland.
Thetransportprocessoflaser generated aerosols influences the detection capabilitiesoflaser ablation microanalysis.Therefore,beside important fac- ESIisapowerfulmethod to analyseproteins and non-covalent complexes in tors like ablation cellvolume,cellgeometry,gas flowpattern [1,2], the the gasphase.The ionisation processleadstothe formation of multiply volume (length) of the transporttube is anotherrelevant factor,which needs charged ions forlarge compounds.The ionisation mechanisms still remain to be investigated. unclear,although thereissome evidence of thevalidity of the Charged
Residue Modeland for the control by apparent gas-phase basicities(GBapp ). Theaim of this study was to investigate the aerosol manipulation in LA- In this work, we present afastand innovative methodology, based on ESSI- ICP-MS. Thesignalintensity changesofvarious elements andthe matrix- MS,todetermine GBapp sofpeptides and proteins at atmospheric pressure. relatedchanges weredetermined and comparedfor different transportsys- Vapor of volatilereference bases was introduced to react with the protein tems.For these measurementsvariablemassloads were introduced to the ions in the atmosphericpressure region, beforethe MS inlet. Thevapor ICP[3].The experimentswerecarriedout using ns and fs laser ablation pressure wasclosetothe saturation pressure,ensuring ahigh collision rate systems and NIST 610, brassstandardreference materials as samples.The with the protein ions and efficient deprotonation reactions.The proof of influentialparametersrelated to the transport system will be discussedin principle was made using bradykinin derivatives,substance Pand insulin this presentation. chainB.Weobtained valuesinexcellent agreement with theGB app values obtained at low pressure (kinetic or bracketing methods). Theseexperiments wereextended to model proteins (unbiquitin, lysozyme [1]D.Bleiner,D.Günther, J. Anal.At. Spectrom., 2001, 16,449. andcytochromec)indenaturingand non-denaturingbuffer. Theresults [2]L.Moenke-Blankenburg, M. Gäckle, D. Günther,J.Kammel, Plasma werecomparedtothe GB calculated with an electrostatic model and con- Source Mass Spectrometry, ed. K. E. Jarvis and A. L. Gray, Royal Society app firm that GBapp control of the protein ionization in the gas phase. Moreover, of Chemistry, Cambridge,UK, 1989 some evidence of thepresence of ionpairing betweenionized basic and [3]I.Kroslakova, D. Günther, J. Anal.At. Spectrom., 2007, 22,51. acidicsitesare given by thesemeasurements.
ESSI-MSoffers the unique possibility to measureGB app of peptides and proteins at atmospheric pressure with good sensitivity (forconcentrations less than 10 μ Mindenaturingornon-denaturingbuffer), verygood accu- racy (less than2%) andinashort time (less than30minutestoscreen up to 23 volatile bases). ANALYTICAL CHEMISTRY426 CHIMIA 2007, 61,No. 7/8
Analytical Chemistry 37 Analytical Chemistry 38
MassLoad Induced MatrixEffectsinLA-ICP-MS:AFurtherInsight Levelsofoxidative stressbiomarkers in workers exposed to Diesel Ex- haustParticulate GiselaFontaine,Detlef Günther Ari Setyan 1 ,Jean-Jacques Sauvain1 ,Michael Riediker 1 ,MichelJ.Rossi2 , ETH Zürich, LaboratoryofInorganic Chemistry, W.-Pauli-Strasse 10, Michel Guillemin1 CH-8093 Zürich,Switzerland,[email protected] 1 Institute of Occupational Health Sciences,Rue du Bugnon 19, CH-1005 Laserablation (LA) is nowadays broadly used as asample introduction Lausanne, Switzerland method forinductively coupled plasma massspectrometry(ICPMS) due to 2 SwissFederal Institute of Technology,Air and Soil Pollution Laboratory, its easysample preparation, fast handling and its potential forhigh spatial CH-1015 Lausanne, Switzerland resolution. However,amount and composition of laser generated aerosols strongly depend on the matrix, especially on the ablation characteristics of TheincreaseofexposuretoPM and PM (particulatematterwith thesample. Differently sized and composed particles can then causefrac- 10 2.5 aerodynamic diameter smaller than 10 μ mand 2.5 μ m, respectively) has tionation by incomplete vaporization,atomizationand ionization of large been found to be associated with arange of adverse health effects.Surface particlesinthe plasma 1 and make the useofcloselymatched ablation condi- characteristics(chemical reactivity,surface area) areofprime importance to tions and calibration standards crucial. This was emphasized by Kroslakova2 understand the mechanisms which lead to harmful effects.Ahypothetical who found amatrix effect induced by the massload of the plasma: higher mechanism to explainthese adverse effectsofparticulatematteristhe abil- plasmaloads result in asubsequent lossofespecially the volatile elements ity of some components (organics,metalions)adsorbed on theseparticles to relative to Calcium, aloweringofthe plasma loadleads to the opposite ef- generate reactive oxygen species(ROS),and thereby to causeoxidative fect. stress in biological systems. ROS can attack almost any cellularstructure, In this study, theseeffects werefurther evaluated. Thefocusofthe mass leading to the formation of awide variety of degradation products which loadstudieswas extendedtodifferent matrices (e.g.the less absorbing canbeusedasabiomarkerofoxidative stress. NIST612, zircon) to study the relation of the sample matrix and itssubse- quent ablation behavior with the degree of mass load bias.Additionally, the Theaim of thepresent research project is to demonstrate an associa- influence of water addition to the ICPwas studied, since wet plasma condi- tion between the exposuretoDiesel ExhaustParticulate (DEP)and the oxi- tions have been found to provide morestable ionization conditions3 .Mass dative stress status.For that purpose, asurvey is conductedinreal occupa- load experimentsweretherefore carried out with a small amount of water tional situations whereworkersare exposed to DEP(bus depots). addedtothe laseraerosol by aPFA microflow nebulizer andwill be dis- cussed. Analytical methods allowing the determination of several biomark- ersofoxidative stress in urine or serumofvolunteershave been validated. [1]H.-R. Kuhn, D. Günther, J. Anal.Atom. Spectrom., 2004,19, 1158- Results about biomarkerslevelsdetermined in such matrix will be presented 1164. anddiscussedfor their applicability andinrelationtothe particulate expo- [2]I.Kroslakova, D. Günther, J. Anal.Atom. Spectrom., 2007,22, 51-62. sure variables. [3]C.J.O'Connor,B.L.Sharp, P. Evans, J. Anal.At. Spectrom. 2006,21, 556-565.
Analytical Chemistry 39 Analytical Chemistry 40
Ultra-fastquantitation of saquinavir in human plasma by MALDI- LA-ICP-MS-aversatile analytical technique in material science SRM ChristianFrei a),Friedrich Waibel b),Detlef Günther a) Michel Wagner, Emmanuel Varesio,GérardHopfgartner a)ETH Zurich, LaboratoryofInorganic Chemistry, 8093 Zurich,Switzerland Life Sciences Mass Spectrometry, b)UmicoreMaterials AG, FL 9596 Balzers, Liechtenstein EPGL,University of Lausanne,University of Geneva, Bd dYvoy 20, 1211 Geneva-4, Switzerland Laserablation inductively coupled plasmamassspectrometry(LA-ICP-MS) is afastand versatile analytical technique dedicated for the analysis of con- Quantitation of low molecular weightcompounds (LMWC) in biological ducting and non-conducting solid materials.Inrecentyears, laser ablation as samplesbylow laserfrequencymatrix-assistedlaser/desorption time-of- sampling technique coupled to ICP-MS wasfrequently applied fortrace flight mass spectrometers (MALDI-TOF) remainsachallenging task.The elementquantification in solid materials of high purity [1].One majorad- hyphenation of MALDIwith triple quadrupole massspectrometers vantage is thatitdoesnot require time consuming sample preparation and (MALDI-QqQ) hasbeen shown to open newopportunitiesinthe quantita- digestion. Therefore, this sampling technique has promising potential for tion of LMWC [1]. quality control of industrial samples. We illustrate herein its potentialinthe selected reaction monitoring mode In this study GeSbTephasechange materials werecharacterized for (MALDI-SRM)using aMDS Sciex MALDI-QqQ prototype equipped with stoichiometryand surface impurities in chromiumweredeterminedafter ahigh repetition (1 KHz)laser for clinical chemistry purposes.Saquinavir, different cleaning procedures.All measurementswerecarried out usinga an anti-HIV drug largely employedinAIDSmultitherapies,istaken for ex- 193 nm ArFExcimer laser system (GeolasPro, CoherentLambda Physik, ample. Göttingen, Germany) equipped with acomputer-controlled stage and a Threeimportant aspectsare highlighted: petrographic microscope. Theappliedinductively coupled plasma mass 1. Thedramatic throughput improvement allows ultra-fast quantitation (less spectrometer was an Agilent 7500ce (Agilent Technologies,Tokyo,Japan). than 10 secondsper sample),over arange of concentrations (5 to 10000 Four different ablation procedures(single pulse, drilling-mode,raster-mode, ng/ml) meeting thosecoveredinclinical trials. andmatrix ablation) were comparedand discussedindetailconcerning their 2. Thespotting processhas been automated with aprocessing station (Shi- capabilitiesand limitations.Using single pulseablation adepth resolution of madzu Xcise),customized to perform electrodeposition. 300-400 nm per pulsewas achieved fordepth profiles in Cr.Due to the uni- 3. Specific experimental designhas been employed to tackle the repro- form particle size distribution andthe constant mass loadinto the plasma duciblity issues of MALDIand arediscussed:a.the useofaninternalstan- over the entireablation period, raster mode showed highestprecisionin dard,b.the rastering mode of the laserbeam and c. repeating the spotting of terms of stoichiometry determinations on the phase change materials using each sample. matrix-matched in-housereference samples forcalibration. Furthermore the sample homogeneity wasinvestigatedcomparing allfour ablation tech- [1]P.Kovarik,C.Grivet, E. Bourgogne, G. Hopfgartner, Rapid Commun. niques. Mass Spectrom ., 2007, 21,911-919 [1]J.S.Becker and H. J. Dietze,Int.J.MassSpectrom., 2003, 228,127 ANALYTICAL CHEMISTRY/MEDICINAL CHEMISTRY427 doi:10.2533/chimia.2007.427 CHIMIA 2007, 61,No. 7/8
Analytical Chemistry 41 Analytical Chemistry 42
Sensor Arrays forthe Analysis of Sugars in Aqueous Solution Investigation of Hypericum species by LC/MS
Friederike Zaubitzer,Kay Severin G. Sibailly,K.Ndjoko,A.Marston, andK.Hostettmann* Institutdes Sciences et Ingénerie Chimiques, Ecole Polytechnique Fédérale LaboratoryofPharmacognosyand Phytochemistry, School of de Lausanne (EPFL),CH-1015 Lausanne, Switzerland Pharmaceutical Sciences,University of Geneva, University of Lausanne, Fluorescence sensors for sugars havereceived enormous interestinrecent Quai Ernest-Ansermet 30,CH-1211 Geneva 4, Switzerland years. Most efforts have focused on the development of sensors with a highly selectivityfor aparticular sugar. This is generally accomplished with thehelp of synthetic receptors, which display ahigh specificity. An interest- Acharacteristic of plant species fromthe genus Hypericum (Hypericaceae) ing alternative is the utilization of asensor array technology. In asensor is the presence of pigments belonging to the class of naphthodianthrones. array, severalnon-specific sensorsare combined and the analyteisthen Theseplants havemany traditional uses and, notably, Hypericum perfora- identified with patternrecognition tools. This technique has successfully tum is employedfor the treatment of mild depression. Severalstudiesdeal been appliedfor different analytical problems [1], but the utilization of sen- with the activitiesofthe numerous constituentsofthe genus or compare sor arrays for sugars is virtually unexplored [2]. We describeasensor array, different Hypericum species [1,2]. Thegenus is alsoreputed forcases of which is based on the reversible coupling of fluorescent hydrazides with the poisoningincattle(hypericism) which alsohave their origin in the presence aldehyde group of reducing sugars. of thesecompounds.Morerecently, the naphthodianthrones have assumed importance forthe photodynamic therapy of cancer. O O N In order to determine the relative contents of hypericin and pseudohypericin Fluorophore NH NH + O Sugar Fluorophore N Sugar 2 H Fluorophore in theseplants,extraction of several species of St.-John's wortwas per- Fig.1.: Schematic representation of afluorescence sensor for sugars formedbydifferent proceduresinordertooptimizethe yield of the active constituents. Discrimination is achieved by exploitation of differences in fluorescence HPLC-UV/DAD and HPLC-MS methods werethen developed forthe emission intensities whichdependonthe nature of the dye-sugar derivate analysis of naphthodianthrones in the plants. It was found that Hypericum and the reaction equilibria in solution. calycinum L. does not contain this classofcompounds.
[1]K.J.Albert, N. S. Lewis, C. L. Schauer, G. A. Sotzing, S. E. Stitzel, [1] FicoG., VitaliniS., Colombo N.,TomeF., Hypericum perforatum L.,H. T.P. Vaid,D.R.Walt, Chem.Rev. 2000,100, 2595. maculatum Crantz. ,H.calycinum L .and H. pulchrum L .: phytochemical [2] J. W. Lee, J.-S. Lee, Y.-T. Chang, Angew. Chem. Int. Ed. 2006,45, andmorphological studies., 2006,1,1129-1132. 6485. [2]OzturkY., Testing the andipressant effects of Hypericum specieson animal models,Pharmacopsychiatry, 1998,31, 37.
Medicinal Chemistry 43 Medicinal Chemistry 44
Thermodynamic and KineticConsiderations of theBinding Processof Novelguanidine-type 5-HT5A receptorantagonists MAG-Antagonists Jens-Uwe Peters ,* Alexander Alanine,AndreAlker,Francesca Blasco, StefanieMesch,DanielStrasser, Morena Spreafico, BrianCutting, Arnulf Dorn, Alain Gast, Luca Gobbi,Sabine Kolczewski, Nicole SachinShelke, Oliver Schwardt,Beat Ernst Kratochwil,Thomas Lübbers, PariMalherbe, Eric Prinssen, Diana Schuhbauer,Lucinda Steward Institute of MolecularPharmacy,University of Basel, Klingelbergstr. 50, 4050 Basel, Switzerland *Discovery Chemistry, F. Hoffmann-La Roche Ltd, CH-4070 Basel
Theinjured adult mammalian central nervous system is an inhibitoryenvi- ronment foraxon regeneration due to specific inhibitoryproteins.The mye- Cl Cl lin-associated glycoprotein (MAG)[1] wasidentified as oneoftheseneurite NH Cl NH NH outgrowth inhibitory proteins [2]. It belongs to the siglec family (sialic-acid N N F H N NH N N bindingimmunoglobulin-like lectin). In earlier studies,weidentified the 2 H F lead structure 1 [3], which was further optimized yielding antagonists with 1 2 3 nM affinities. K i (5-HT 5A)=160 nM K i (5-HT 5A)=2nM K i (5-HT 5A)=3nM screening hit brain/plasma ~0.2 brain/plasma ~4 Cl H N OH COOH Theexpression of the 5-HT receptor in the limbic brain areas suggestsa OH 5A potential role in the modulation of psychiatric diseases. 1 Howeveruntil re- O O O AcHN cently, no selective 5-HT5A receptor ligands were available to study its HO pharmacology in detail. We screened the Roche compound librarytoiden- tifyselective antagonists forthis target, and found several guanidines such 1 as 1 among the mostselective compounds.Asystematic exploration of small substituents (Cl, Me,MeO,F)around the core structureled to 2 with
Thekinetic and thermodynamic propertiesofthese high affinity ligands potent 5-HT5A antagonistic affinity in vitro ,and improved selectivity, apart wereelucidatedbyBiacorestudies.Inaddition, the binding mode wasex- from5-HT 7 .Compound 2 had good PK properties, however alow brain- amined through STDNMR experiments and docking studies. plasma ratio. Thebrain penetration was improved by the introduction of electron-withdrawing substituents,which afforded acompound with in- [1]Quarles, R. H., J. Neurochem. 2007, 100,1431. creased lipophilicity,and reduced basicity, 3 .The series refinement and [2]Crocker,R.H., Curr.Opin. Struct.Biol. 2002, 12,609. structure activity relationship elucidatedinprogressing from the initialhit 1 [3]Shelke, S.,Gao,G-P., Mesch, S.,Gäthje, H.,Kelm, S.,Schwardt, O., to lead compound 3 will be furtherdescribed in the presentation. Ernst, B., Bioorg. Med. Chem, in press . [1]Thomas,D.R. Pharmacol.Ther. 2006, 111(3), 707-714.