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Methods in Enzymology, Vol. 369: Combinatorial Chemistry, Part B PDF

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Preview Methods in Enzymology, Vol. 369: Combinatorial Chemistry, Part B

Preface Combinatorial chemistry has matured from a field where efforts initially focused on peptide-based research to become an indispensable research tool formolecularrecognition,chemical-propertyoptimization,anddrugdiscovery. Originallyusedasamethodtoprimarilygeneratelargenumbersofmolecules, combinatorial chemistry has been significantly influenced and integrated with other important fields such as medicinal chemistry, analytical chemistry, syn- theticchemistry,robotics,andcomputationalchemistry. Eventhoughtheinitialfocusofattentionwasprovidinglargernumbersof molecules with a ‘‘diversity’’ goal in mind, other factors came into play dependingupontheproblemscientistsweretryingtosolve,suchasbioactivity, solubility, permeability properties, PK, ADME, toxicity, and patentability. One can think of combinatorial chemistry and compound screening as an iterative Darwinian process of divergence and selection. Particularly in drug discovery, where time is a critical factor to success, combinatorial chemistry offersthemeanstotestmoremoleculehypothesesinparallel. We will always be limited to a finite number of molecules that we can economically synthesize and evaluate. Even with all the advances in automa- tiontechnologies,combinatorialchemistry,andhigher-throughputscreensthat improve our ability to rapidly confirm or disprove hypotheses, the synthesis and screening cycle remains the rate-determining process. Fortunately, we continue to make great strides forward in the quality and refinement of pre- dictivealgorithmsandinthebreadthofthetrainingsetsamassedtoaidinthe drugdiscovery/compoundoptimizationiterativeprocess. Anyonewhohasoptimizedchemicalreactionsforcombinatoriallibraries orprocesschemistryknowsfirsthandhowmuchexperimentationisrequiredto identify optimal conditions. Chemical feasibility is at the heart of small mol- ecule discovery and chemotype prioritization since it essentially defines what canandcannotbeanaloged(i.e.,analogability).Althoughanalogabilityisnot the only driving factor, quite often it is overlooked. For example, when com- mercially-available compounds or complex natural products are screened, the leads generated are often dropped because of the difficulty to rapidly analog themintheleadoptimizationstage. The desirability of a chemotype is a function of drug-likeness, potency, novelty, and analogability. A particularly attractive feature of combinatorial chemistry is that when desirable properties are identified, they can often be xiii xiv preface optimized through second-generation libraries following optimized synthetic protocols. If this process of exploring truly synthetically accessible chemical spaces could be automated, then it would open up the exciting possibility of modelingtheiterativesynthesisandscreeningcycle. Predicting, or even just mapping, synthetic feasibility is a sleeping giant; fewpeoplearelooking intoit,andtheramificationsofabreakthroughwould berevolutionaryforbothchemistryanddrugdiscovery.In-roadstopredicting (orevenjustmapping)chemicalfeasibilityhavethepotentialtohaveaslarge an impact on drug discovery as computational models of bioavailability and drugability.Theseareimportantquestionswherescientistsarenowstartingto generate a large-enough body of information on high-throughput synthetic chemistry to begin to more globally understand what is cost-effectively pos- sible. Within the biopharmaceutical industry, significant investments in new technologieshavebeenmadeinmolecularbiology,genomics,andproteomics. However, with the exception of combinatorial chemistry, relatively little has been done to advance the fundamental nature of chemistry in drug discovery fromaconceptualperspective. Now, after having gone through the molecule-generating period where research institutions have a large historical compound collection and the pro- liferation of combinatorial chemistry services, the trend is now after making more targeted-oriented molecular entities also known as ‘‘focused libraries.’’ An important emerging question is: How can one most effectively make the best possible ‘‘focused libraries’’ to answer very specific research questions, givenallthepossiblemoleculesonecouldtheoreticallysynthesize? The first installment in this series (Volume 267, 1996) mostly covered peptide and peptidomimetic based research with just a few examples of small molecule libraries. In this volume we have compiled cutting-edge research in combinatorial chemistry, including divergent areas such as novel analytical techniques, microwave-assisted synthesis, novel linkers, and synthetic ap- proachesinbothsolid-phaseandpolymer-assistedsynthesisofpeptides,small molecules, and heterocyclic systems, as well as the application of these tech- nologiestooptimizemolecularpropertiesofscientificandcommercialinterest. Guillermo A. Morales Barry A. Bunin METHODS IN ENZYMOLOGY EDITORS-IN-CHIEF John N. Abelson Melvin I. Simon DIVISIONOFBIOLOGY CALIFORNIAINSTITUTEOFTECHNOLOGY PASADENA,CALIFORNIA FOUNDING EDITORS Sidney P. Colowick and Nathan O. Kaplan Contributors to Volume 369 Articlenumbersareinparenthesesandfollowingthenamesofcontributors. Affiliationslistedarecurrent. Fernando Albericio (2), University BalanChenera(24),AmgenInc.,Depart- of Barcelona, Barcelona Biomedical mentofSmallMoleculeDrugDiscovery, Research Institute, Barcelona Science One Amgen Center Drive, Thousand Park, Josep Samitier 1, Barcelona, Oaks,California,91320 08028,Spain James W. Christensen (5), Advanced Alessandra Bartolozzi (19), Surface ChemTech Inc., 5609 Fern Valley Road, Logix, Inc., 50 Soldiers Field Place, Louisville,Kentucky,40228 Brighton,Massachusetts,02135 Andrew P. Combs (12), Incyte Corpo- Hugues Bienayme´ (24), Chrysalon Mo- ration,Wilmington,Delaware,19880-0500 lecularResearch,IRC,11AlbertEinstein Avenue,Villeurbannem,69100,France Scott M. Cowell (16), Department of Chemistry, University of Arizona, Sylvie E. Blondelle (18), Torrey Pines Tucson,Arizona,85721 Institute for Molecular Studies, 3550 General Atomics Court, San Diego, Stefan Dahmen (7), Institut fur Orga- California,92121 nische Chemie, RWTH Aachen, Pirlet- Ce´sar Boggiano (18), Torrey Pines Str.1,Aachen,52074,Germany Institute for Molecular Studies, 3550 Ninh Doan (17), Division of Hematology General Atomics Court, San Diego, and Oncology, Department of Internal California,92121 Medicine,UCDavisCancerCenter,Uni- Stefan Bra¨se (7), Institut fu¨r Organische versity of California Davis, Sacramento, Chemie, Universita¨t Karlsruhe (TH), California,95817 Fritz-Haber-Weg 6, Karlsruhe, D-76131, Roland E. Dolle (8), Senior Director of Germany Chemistry, Department of Chemistry, Andrew M. Bray (3), Mimotopes Pty Adolor Corporation, 700 Pennsylvania Ltd., 11 Duerdin Street, Clayton, Vic- Drive,Exton,Pennsylvania,19345 toria,3168,Australia Nicholas Drinnan (14), Alchemia Pty Wolfgang K.-D. Brill (23), Discovery Ltd., Eight Mile Plains, Queensland Research Oncology, Pharmacia Italy 4113,Australia S.p.A,VialePasteur10,Nerviano(MI), I-20014,Italy Amanda M. Enstrom (17), Division of Hematology and Oncology, Department MaxBroadhurst(14),AlchemiaPtyLtd., of Internal Medicine, UC Davis Cancer EightMilePlains,Queensland4113,Aus- Center, University of California Davis, tralia Sacramento,California,95817 ix x contributors to volume 369 Liling Fang (1), ChemRx Division, Dis- RichardHoughten(25),TorreyPinesIn- coveryPartnersInternational,385Oyster stitute for Molecular Studies, 3550 Gen- Point Boulevard, Suite 1, South San eral Atomics Court, Room 2-136, San Francisco,California,94080 Diego,California,92121 Eduard R. Felder (23), Discovery Re- Victor J. Hruby (16), Department of search Oncology, Pharmacia Italy Chemistry, University of Arizona, S.p.A., Viale Pasteur 10, Nerviano Tucson,Arizona,85721 (MI),I-20014,Italy ChristopherHulme(24),AmgenInc.,De- A´rpa´d Furka (5), Eo¨tvo¨s Lora´nd Univer- partmentofSmallMoleculeDrugDiscov- sity, Department of Organic Chemistry, ery, One Amgen Center Drive, 29-1-B, P.O. Box 32, Budapest 112, H-1518, ThousandOaks,California,91320 Hungary SharonA.Jackson(12),AventisPharma- A.Ganesan(22),UniversityofSouthamp- ceuticals, 202-206, Bridgewater, New ton,DepartmentofChemistry,Highfield, Jersey,08807-0800 Southampton,SO171BJ,UnitedKingdom IanW.James(3),MimotopesPtyLtd.,11 J. Gabriel Garcia (20), 4SC AG, Am Duerdin Street, Clayton, Victoria, 3168, Klopferspitz 19A, 82152, Martinsried, Australia Germany Wyeth Jones (24), Amgen Inc., Depart- Brian Glass (13), Incyte Corporation, mentofSmallMoleculeDrugDiscovery, Wilmington,Delaware,19880-0500 OneAmgenCenterDrive,29-1-B,Thou- sandOaks,California,91320 MatthiasGrathwohl(14),AlchemiaPty Ltd.,EightMilePlains,Queenland4113, Patrick Jouin (10), CNRS UPR 9023, Australia CCIPE,141,ruedelaCardonille,Mont- pellierCedex05,34094,France Michael J. Grogan (19), Surface Logix, Inc., 50 Soldiers Field Place, Brighton, C.OliverKappe(11),InstituteofChemis- Massachusetts,02135 try, Karl-Franzens-University Graz, Heinrichstrasse28,Graz,A-8010,Austria Xuyuan Gu (16), Department of Chemistry, University of Arizona, StevenA.Kates(19),SurfaceLogix,Inc., Tuscon,Arizona,85721 50SoldiersFieldPlace,Brighton,Massa- chusetts,02135 EricHealy(5),AdvancedChemTechInc., 5609 Fern Valley Road, Louisville, Viktor Krchnˇa´k (6), Torviq, 3251 West Kentucky,40228 LambertLane,Tuscon,Arizona,85742 Timothy F. Herpin (4), Rhoˆne-Poulenec KitS.Lam(15,17), DivisionofHematol- Rorer, 500 Arcola Road, Collegeville, ogy and Oncology, Department of In- Pennsylvania,19426 ternal Medicine, UC Davis Cancer Center, University of California Davis, CorneliaE.Hoesl(25),TorreyPinesIn- Sacramento,California,95817 stitute,Room2-136,3550GeneralAtom- icsCourt,SanDiego,California,92121 AlanL.Lehman(17), DivisionofHema- tology and Oncology, Department of In- ChristopherP.Holmes(9),AffymaxInc., ternal Medicine, UC Davis Cancer 4001 Miranda Avenue, Palo Alto, Center, University of California Davis, California,94304 Sacramento,California,95817 contributors to volume369 xi RuiwuLiu(15,17), DivisionofHematol- E.R. Palmacci (13), 77 Massachusetts ogy and Oncology, Department of In- Avenue,T18-209,Cambridge,Massachu- ternal Medicine, UC Davis Cancer setts,02139 Center, University of California Davis, Sacramento,California,95817 Yijun Pan (9), Affymax Inc., 4001 Mi- randa Avenue, Palo Alto, California, MatthiasLormann(7),Kekule´-Institutfu¨r 94304 Organische Chemie und Biochemie der Rheinischen, Friedrich Wilhelms Univer- JackG.Parsons(3),MimotopesPtyLtd., sita¨t Bonn, Gerhard-Domagk-Strasse 1, 11 Duerdin Street, Clayton, Victoria, Bonn,D-53121,Germany 3168,Australia Jan Marik (15), Division of Hematology Robert Pascal (10), UMR 5073, Univer- and Oncology, Department of Internal site´ de Montpellier 2, CC017, place Medicine,UCDavisCancerCenter,Uni- Euge`ne Bataillon, Montpellier Cedex 05, versity of California Davis, Sacramento, F-34094,France California,95817 Clemencia Pinilla (18), Torrey Pines In- Katia Martina (23), Discovery Research stituteforMolecularStudiesandMixture Oncology, Pharmacia Italy S.p.A., Viale Sciences, Inc., 3550 General Atomics Pasteur10,Nerviano(MI),I-20014,Italy Court,SanDiego,California,92121 JoesephMaxwell(17),DivisionofHema- Obadiah J. Plante (13), Massachusetts tology and Oncology, Department of In- Institute of Technology, Department of ternal Medicine, UC Davis Cancer Chemistry, 77 Massachusetts Avenue, Center, University of California Davis, Cambridge,Massachusetts,02139-4307 Sacramento,California,95817 Gregory Qushair (2), University WimMeutermans(14),AlchemiaPtyLtd., of Barcelona, Barcelona Biomedical 3 Hi-Tech Court, Brisbane Technology Research Institute, Barcelona Science Park,EightMilePlains,QLD4113,Aus- Park, Josep Samitier 1, Barcelona, tralia 08028,Spain George C. Morton (4), Rhoˆne-Poulenc JorgRademann(21),Eberhard-Karls-Uni- Rorer, 500 Arcola Road, Collegeville, versity, Tu¨bingen, Institute of Organic Pennsylvania,19426 Chemistry,AufderMorgenstelle18,Tu¨- AdelNefzi(25),TorreyPinesInstitutefor bingen,72076,Germany MolecularStudies,3550GeneralAtomics Court,SanDiego,California,92121 Joseph M. Salvino (8), Director of Com- binational Chemistry, Adolor Corpor- Thomas Nixey (24), Amgen Inc., Depart- ation, 700 Pennsylvania Drive, Exton, mentofSmallMoleculeDrugDiscovery, Pennsylvania,19345 OneAmgenCenterDrive,29-1-B,Thou- sandOaks,California,91320 Peter H. Seeberger (13), Laboratorium fuer Organische Chemie, HCI F 315, JohnM.Ostresh(25),TorreyPinesInsti- Wolfgang-Pauli-Str.10,ETH-Hoengger- tute,Room2-136,3550GeneralAtomics berg,CH-8093Zu¨rich,Switzerland Court,SanDiego,California92121 Craig S. Sheehan (3), Mimotopes Pty VitecekPadeˇra(6),Torvic,3251WLam- Ltd., 11 Duerdin Street, Clayton, Vic- bertLane,Tucson,Arizona,84742 toria,3168,Australia xii contributors to volume 369 AdrianL.Smith(24),AmgenInc.,Depart- Jesus Vazquez (2), University of Barce- mentofSmallMoleculeDrugDiscovery, lona, Barcelona Biomedical Research One Amgen Center Drive, Thousand Institute, Barcelona Science Park, Josep Oaks,California,91320 Samitier1,Barcelona,08028,Spain Re´gine Sola (10), UMR 5076, Ecole MichaelL.West(14),AlchemiaPtyLtd., Nationale Supe´rieure de Chimie de Eight Mile Plains, Queensland 4113, Montpellier, 8, rue Delaware l’Ecole Australia Normale, Montpellier Cedex 05, F- 34296,France Zemin Wu (3), Mimotopes Pty Ltd., 11 AiminSong(17),UniversityofCalifornia, Duerdin Street, Clayton, Victoria, 3168, UC Davis Cancer Center, Division of Australia Hematology and Oncology, 4501 X Street,Sacramento,California,95817 BingYan(1),ChemRxDivision,Discovery PartnersInternational,385OysterPoint, Alexander Stadler (11), Institute of Boulevard,Suite1,SouthSanFrancisco, Chemistry, Karl-Franzens-University California,94080 Graz, Heinrichstrasse 28, Graz, A-8010, Austria YongpingYu(25),TorreyPinesInstitute, Paul Tempest (24), Amgen Inc., Depart- Room 2-136, 3550 General Atomics mentofSmallMoleculeDrugDiscovery, Court,SanDiego,California,92121 OneAmgenCenterDrive,29-1-B,Thou- sandOaks,California,91320 Florencio Zaragoza (26), Medicinal Chemistry,NovoNordiskA/S,NovoNor- David Tumelty (9), Affymax, Inc., diskPark,Malov,2760,Denmark 4001 Miranda Avenue, Palo Alto, California,94304 JiangZhao(1),ChemRxDivision,Discov- Josef Vagner (16), Department of Chem- ery Partners International, 385 Oyster istry,UniversityofArizona,Tuscon,Ari- Point Boulevard, Suite 1, South San zona,85741 Francisco,California,94080 [1] high-throughput LC/UV/MSanalysis of libraries 3 [1] High-Throughput Parallel LC/UV/MS Analysis of Combinatorial Libraries By Liling Fang, Jiang Zhao, and Bing Yan Introduction Combinatorial chemistry and high-throughput organic synthesis allow thepreparationofalargenumberofdiversecompoundsinarelativeshort period of time in order to accelerate discovery efforts in the pharmaceut- ical and other industries. A library can comprise hundreds to thousands ofcompounds with theneedtorapidlyanalyzethose compoundsfortheir identityandpurity.Differentcompoundseparationandmassspectrometry (MS)techniqueshavebeenappliedforthecharacterizationofcombinator- iallibraries.Theseincludeseparationtechniquessuchasliquidchromatog- raphy(LC)andcapillaryelectrophoresisanddifferent ionizationmethods andmassanalyzers.1–3LC/MS*isthemostpopulartechniqueusedincom- binatorial library analysis because it combines separation, molecular weight determination,andrelativepurityevaluation inasinglesamplein- jection. However, the throughput of conventional LC/MS could not meet the need to analyze every member in a large combinatorial library in a timely fashion. Higher-throughput analysis was achieved by utilizing shorter columns at higher flow rates.4 Supercritical fluid chromatography (SFC)/MS has 1A. Hauser-Fang and P. Vouros, ‘‘Analytical Techniques in Combinatorial Chemistry’’ (M.E.Swartz,ed.).MarcelDekker,NewYork,2000. 2B.Yan,‘‘AnalyticalMethodsinCombinatorialChemistry.’’Technomic,Lancaster,2000. 3D.G.Schmid,P.Grosche,H.Bandel,andG.Jung,Biotechnol.Bioeng.Comb.Chem.71, 149(2001). *Abbreviations:CLND,chemiluminescencenitrogendetection;ClogP,calculatedpartition coefficient; ELSD, evaporative light scattering detection; ESI-MS, electrospray ionization mass spectrometry; FWHM, full width at half maximum; i.d., inner diameter; LC, HPLC, liquid chromatography, high-performance liquid chromatography; LC/MS, liquid chroma- tography – mass spectrometry; LC/MS/MS, liquid chromatography – mass spectrometry – mass spectrometry; LC/UV/MS, liquid chromatography mass spectrometry with a UV detector; LIB, compound library; log P, water/octanol partition coefficient; MUX, multiplexed; RSD, relative standard deviation; SFC, supercritical fluid chromatography; TFA,trifluoroaceticacid;TIC,totalioncurrent;TOF,timeofflight;TOFMS,timeofflight massspectrometry. 4H. Lee, L. Li, and J. Kyranos, Proceedings of the 47th ASMS Conference on Mass SpectrometryandAlliedTopics,Dallas,Texas,June13–17,1999. Copyright2003,ElsevierInc. Allrightsreserved. METHODSINENZYMOLOGY,VOL.369 0076-6879/03$35.00 4 analytical techniques [1] beenusedtoachievedesirablehighspeedtakingadvantageofthelowvis- cosityofCO .5However,theserialLC/MSapproachbyitsnaturedoesnot 2 match the speed of parallel synthesis. Parallel LC/MS is the method of choiceto increasethroughputwhilemaintainingthe separationefficiency. An eight-probe Gilson 215/889 autosampler was incorporated into a quadruple mass spectrometer.6 This arrangement enabled the injection of eight samples (a column from a 96-well microtiter plate) simultaneously for flow-injection analysis/MS (FIA-MS) analysis to achieve a throughput of 8 samples/min. A novel multiplexed electrospray interface (MUX)7 was developed in 1999 and became commercially available for parallel high-throughput LC/UV/MS analysis. The eight-way MUX consists of eight nebulization-assisted electrospray ionization sprayers, a desolvation gas heater probe, and a rotating aperture. It can accommodate all eight high-performanceliquidchromatograph(HPLC)streamsatareducedflow rateof<100 (cid:2)l/minperstreamandconductelectrosprayionizationforall eight streams simultaneously. Ions are continuously formed at the tip of eachsprayerandtheMUXinterfaceallowssprayerstobesampledsequen- tiallyusingtherotatingaperturedrivenbyaprogrammablesteppermotor. At any given time, only ions from one stream are admitted into the ion samplingcone,whileionsfromtheothersevensprayersareshielded.Each liquidstreamissampledforapresettimewithmassspectraacquiredinfull massrangeintoeightsimultaneouslyopendatafilessynchronizedwiththe spray being sampled. With a 0.1-s acquisition time per sprayer and 0.05-s intersprayerdelaytime,thetime-of-flight(TOF)massanalyzercanacquire a discrete data file of electrospray ion current sampled from each stream over the entire HPLC separation with a cycle time of 1.2 s. Therefore, this eight-way MUX-LCT was like having eight individual electrospray ionization (ESI)-MS systems working simultaneously. The MUX interfaceenables the coupling of parallel liquidchromatog- raphy to a single mass spectrometer. This technology has had a great impact in high-throughput LC/MS analysis. In drug development, a four- way MUX interface was used on a triple quadrupole mass spectrometer to simultaneously validate LC/MS/MS methods for the quantitation of loratadine and its metabolite in four different biological matrixes8 and of 5M.C.Ventura,W.P.Farrell,C.M.Aurigemma,andM.J.Greig,Anal.Chem.71,2410 (1999). 6T.Wang,L.Zeng,T.Strader,L.Burton,andD.B.Kassel,RapidCommun.MassSpectrom. 12,1123(1998). 7V.DeBiasil,N.Haskins,A.Organ,R.Bateman,K.Giles,andS.Jarvis,RapidCommun. MassSpectrom.13,1165(1999). 8M.K.Bayliss,D.Little,D.M.Mallett,andR.S.Plumb,RapidCommun.MassSpectrom. 14,2039(2000). [1] high-throughput LC/UV/MSanalysis of libraries 5 diazepaminratlivermicrosomesforinvitrometabolicstability.9Thefour- channel LC/MS/MS system was also reported for the quantification of a druginplasmaonboththenarrow-boreandcapillaryscales.10Byincorpor- ating divert valves into this system, aliquots of plasma could be directly analyzedwithoutsamplepreparation.Thefour-channelLC/MS/MShasre- ducedmethodvalidationtime,increasedsamplethroughputby4-fold,and afforded adequate sensitivity, precision, and negligible intersprayer cross- talk.8,9 In protein analysis, an eight-way MUX coupled with a TOFMS analyzer has proved to be a powerful tool to monitor the protein purifica- tionprocessbyscreeningfractionsfrompreparativeion-exchangechroma- tography with a throughput of 50 protein-containing fractions in less than anhour.11 Ahigh-pressuregradientparallelpumpingsystem(JASCOPAR-1500) hasbeendevelopedtoconducthigh-throughputparallelliquidchromatog- raphy.12 It is a 10-pump system where two pumps are used to generate a binary gradient and eight pumps to deliver the mixed solvent to eight LC columns.Comparingthissystemtoaconventionalsystemwithtwopumps orabinarypumpforLCgradientandasimplesplittertodividethegradi- enttoeightLCcolumns,thissystemcanensureuniformflowratesthrough eachLCcolumn.Thissystemhasbeenusedforpeptidesandcombinatorial sample,12 protein analysis,13 and bioanalysis.9 We have optimized an eight-way MUX coupled to a TOFMS analyzer to carry out eight-channel parallel LC/UV/MS analysis of combinatorial libraries14 in the past 2 years. This system has not only provided the capacity needed for library analysis, but also enabled simultaneous evalu- ation of experimental parameters to expedite the method development process. In this chapter, we discuss the optimization of this system and present a high-throughput protocol for combinatorial library analysis. We also compare the eight-channel parallel LC/UV/MS system to a conven- tional single channel LC/UV/MS system in terms of performance and operation. 9D.Morrison,A.E.Davis,andA.P.Watt,Anal.Chem.74,1896(2002). 10L.Yang,T.D.Mann,D.Little,N.Wu,R.P.Clement,andP.J.Rudewicz,Anal.Chem.73, 1740(2001). 11B.Feng,A.Patel,P.M.Keller,andJ.R.Slemmon,RapidCommun.MassSpectrom.15, 821(2001). 12D.Tolson,A.Organ,andA.Shah,RapidCommun.MassSpectrom.15,1244(2001). 13B. Feng, M. S. McQueney, T. M. Mezzasalma, and J. R. Slemmon, Anal. Chem. 73, 5691(2001). 14J. Zhao, D. Liu, J. Wheatley, L. Fang, and B. Yan, Proceedings of the 49th ASMS ConferenceonMassSpectrometryandAlliedTopics,Chicago,IL,May,27–31,2001.

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